1

DEPARTMENT OF HEALTH AND HUMAN SERVICES

FOOD AND DRUG ADMINISTRATION

CENTER FOR FOOD SAFETY AND APPLIED NUTRITION

FOOD ADVISORY COMMITTEE MEETING

Advice on CFSAN'S Draft Report:

Approaches to Establish Thresholds for Major Food Allergens and for Gluten in Food

Thursday, July 14, 2005

8:30 A.M. to 5:20 P.M.

Greenbelt Marriott

6400 Ivy Lane

Grand Ballroom

Greenbelt, Maryland 20770

P A R T I C I P A N T S

FOOD ADVISORY COMMITTEE STANDING MEMBERS:

Richard A. Durst, Ph.D. - Acting Chairman

Jeffrey A. Barach, Ph.D. (Industry Representative)

Patrick S. Callery, Ph.D.

Dennis Gonsalves, Ph.D., M.S.

Jean M. Halloran (Consumer Representative)

Douglas C. Heimburger, M.D., M.S.

Margaret C. McBride, M.D.

Mark Nelson, Ph.D. (Industry Representative)

Carol I. Waslien Ghazaii, Ph.D., R.D.

TEMPORARY VOTING MEMBERS:

Petr Bocek, M.D., Ph.D. (Absent 7/14/05 Session)

Margaret Briley, Ph.D., R.D.

Erica Brittain, Ph.D.

Ciaran P. Kelly, M.D.

Soheila June Maleki, Ph.D.

David O. Oryang

Marc D. Silverstein, M.D.

Suzanne Teuber, M.D.

FOOD AND DRUG ADMINISTRATION:

Robert E. Brackett, Ph.D. - Director

Food and Drug Administration, CFSAN

Catherine Copp, J.D. - Senior Policy Advisor

Food and Drug Administration, CFSAN

Steven M. Gendel, Ph.D. - Senior Scientist

Food and Drug Administration

National Center for Food Safety and Technology

Rhonda Kane, M.S., R.D. - Consumer Officer

Food and Drug Administration, CFSAN

Marcia Moore, Food Advisory Committee, Executive Secretary

P A R T I C I P A N T S (Continued)

FOOD AND DRUG ADMINISTRATION STAFF:

Michael M. Landa, J.D. - Deputy Director for Regulatory Affairs

Food and Drug Administration, CFSAN

Stafano Luccioli, M.D. - Senior Medical Advisor

Food and Drug Administration, CFSAN

GUEST SPEAKERS:

Pekka Collin, M.D., M.P.H. - Professor

University of Tampere, Medical School, Finland

Catherine L. Copp, J.D.

Policy Advisor, CFSAN, FDA

Alessio Fasano, M.D. - Professor of Pediatrics

Medicine & Physiology and Director, the Mucosal

Biology Research Center, Center for Celiac

Research, University of Maryland School of

Medicine

Steven M. Gendel, Ph.D. - Senior Scientist

National Center for Food Safety and Technology, FDA

Rhonda R. Kane, M.S., R.D. - Consumer Safety

Officer CFSAN, FDA

Donald Kasarda, Ph.D. - Consultant and Retired

Senior Scientist, Agriculture Research Service,

USDA

Cynthia Kupper, R.D., C.D. - Executive Director

Gluten Intolerance Group of North America

Joseph A. Murray, M.D. - Professor of Medicine

The Mayo Clinic of Rochester, Minnesota

C O N T E N T S

PAGE

Call to Order and Welcome and Introductions

Richard Durst, Ph.D., Acting Chairman 6

Use of Gluten Thresholds

Catherine L. Copp, J.D., CFSAN, FDA 9

Introduction to Celiac Disease

Joseph Murray, M.D. 11

Patient Perspectives on Celiac Disease

Cynthia Kupper, R.D., C.D. 69

Grains

Donald Kasarda, Ph.D., USDA 84

Question and Answer Session 108

Prospective Studies

Alessio Fasano, M.D. 114

Question and Answer Session 146

Retrospective Studies

Pekka Collin, M.D., M.P.H. 161

Question and Answer Session 182

International Perspectives on Gluten-Free

Rhonda S. Kane, M.S., R.D., CFSAN, FDA 186

Question and Answer Session 198

Public Comments:

Elaine Monarch 212

Alice Bast 220

Mary Schluckabeer 225

Tom P. Sullivan 232

Steve Taylor 240

C O N T E N T S

PAGE

Overview of Approaches to Establishing

Thresholds: Gluten

Steven M. Gendel, Ph.D. 253

Question and Answer Session 257

Committee Discussion:

Panel Discussion with Guest Speakers 259

Gluten and Celiac Disease - FDA Questions 287

Revisit Food Allergens 354

Adjournment

Richard Durst, Ph.D., Acting Chairman 363

P R O C E E D I N G S

CALL TO ORDER AND WELCOME AND INTRODUCTIONS

CHAIRMAN DURST: Good morning. I would

like to call the meeting to order. All right, I

would like to welcome everyone back and also

welcome new participants in our meeting this

morning.

For those of you who weren't here

yesterday, there is a "Conflict of Interest

Statement" over on the table, if you want to refer

to that at all, otherwise I would also ask again

that maybe our participants or our members of the

Food Advisory Committee would introduce themselves

again for the benefit of those who were not here

yesterday.

I am Dick Durst, professor [of]chemistry at

Food Science and Technology Department at Cornell

University.

Marc, would you start it off?

DR. SILVERSTEIN: Marc Silverstein, I'm a

general internist and geriatrician at Baylor Health

Care System in Dallas.

DR. TEUBER: Suzanne Teuber, I am an

allergist at UC-Davis.

MR. ORYANG: I am David Oryang. I am a

risk analyst and agricultural engineer at the

United States Department of Agriculture, Animal and

Plant Health Inspection Service.

DR. KELLY: Good morning. Ciaran Kelly, I

am a gastroenterologist at Harvard Medical School

in Boston.

DR. MALEKI: I am Soheila Maleki. I am a

scientist with the USDA.

DR. BRITTAIN: Erica Brittain, I am a

statistician at the National Institute of Allergy

and Infectious Disease.

DR. BRILEY: Margaret Briley, University

of Texas at Austin, nutritionist.

MRS. MOORE: Marcia Moore, I am the

executive secretary for the Food Advisory Committee

and the Food and Drug Administration.

DR. WASLIEN: I am Carol Waslien,

nutritional epidemiologist at the School of

Medicine, the University of Hawaii.

DR. BRILEY: I am Margaret McBride, child

neurologist at Akron Children's Hospital.

DR. CALLERY: Pat Callery, West Virginia

University, pharmaceutical scientist.

DR. GONSALVES: I am Dennis Gonsalves, a

scientist at USDA.

DR. HEIMBURGER: I am Doug Heimburger, a

physician and nutrition specialist at the

University of Alabama at Birmingham.

DR. BARACH: Jeff Barach with Food

Products Association here, in Washington, D.C., in

regulatory affairs.

DR. NELSON: Mark Nelson with the Grocery

Manufacturers Association here, in Washington,

D.C., and I am responsible for scientific and

regulatory policy.

MS. HALLORAN: Jean Halloran with

Consumers Union located in Yonkers, New York, and I

am director of food policy initiatives.

CHAIRMAN DURST: Thank you very much.

I would also like to remind everyone and

also for our new people here at the meeting that

the "Charge" of the Committee is written out on the

meeting document. The most important thing is that

we are focusing on the "Threshold Working Group

Draft Report on Approaches to Establish Thresholds

for Major Food Allergens and for Gluten in Food."

We are not here to set any kind of

thresholds or discuss the labeling of these foods

for allergens, but strictly to make comments on the

best approaches to use for setting these

thresholds.

Did I cover everything that we need to?

(No verbal response.)

CHAIRMAN DURST: In that case, let's begin

with our first presentation. This is

Catherine Copp, the policy advisor for CFSAN, FDA,

on the use of gluten thresholds.

USE OF GLUTEN THRESHOLDS

MS. COPP: I have been asked this morning

to proceed with discussion on gluten and threshold

levels for gluten or possible thresholds for gluten

framework. It is similar to yesterday. I simply

want to provide you with some context for the

evaluating the Draft Report portion that addresses

gluten in food. This is the hazard of being first.

(Slide.)

MS. COPP: Yesterday, I mentioned the Food

Allergen Labeling and Consumer Protection Act.

This is a new law that Congress passed last August.

Although it focuses primarily on allergens, food

allergens, Congress also directed FDA to address

the separate problem of gluten in food.

When I say directed, I mean that Congress

has mandated that the Agency consider and then

establish regulations according to a schedule to

define "gluten-free" for use on food labels and

also to identify the appropriate use of the term.

As with Allergens, for consumers with

celiac disease, strict avoidance of gluten at

levels that will elicit an adverse effect is the

only means to prevent potentially serious

reactions.

Accurate, complete and informative

labeling on foods can help these consumers achieve

their goal. We believe that understanding

thresholds for gluten and having a sound scientific

framework for evaluating the existence of such

thresholds will help FDA develop a definition of

gluten-free and identify appropriate use of the

term. That's it.

Thank you.

CHAIRMAN DURST: Does anyone have any

comments or question on Catherine's presentation?

(No verbal response.)

CHAIRMAN DURST: Okay. We will move on

then to the presentation from Dr. Joseph Murray,

professor of medicine at the Mayo Clinic of

Rochester, Minnesota, on the introduction to celiac

disease.

INTRODUCTION TO CELIAC DISEASE

DR. MURRAY: Good morning, Committee

Members. I will be providing a general overview to

celiac disease.

(Slide.)

DR. MURRAY: First of all, we will discuss

what is celiac disease. We will discuss, briefly,

the pathogenesis of the disease; who gets it; what

the treatment is, at least in a very relatively

superficial fashion. We will discuss some of the

complications and compliance issues of celiac

disease and a prognosis or future of celiac

disease.

(Slide.)

DR. MURRAY: Obviously, yesterday was

focused on food allergies. "Celiac disease" is one

of the food intolerances that is immune-mediated,

though it is not thought to be IgE-mediated; so, it

comes into the non-IgE-mediated food intolerances

that are mediated by an immune response.

(Slide.)

DR. MURRAY: Where does it happen? It

happens within the smaller intestine, predominantly

the proximal, smaller intestine is the workhorse of

the digestive system. It is this surface of the

intestinal lining that is maximally expanded by the

development of circular folds and on top of these

circular folds the so-called "villi," these villi,

shown here in a histological picture, which

maximize the digestive surface area.

It is on the surface entrocytes of these

villi that most of the enzymes and in the layer

immediately above that in the lumen where most

digestion of the macromolecules from nutrition are

broken down and then absorbed. This is just a

picture. It looks like one of those shag-ply

carpets from the 1970s. This is a normal

appearance.

(Slide.)

DR. MURRAY: However, celiac disease is an

inflammatory state of the small intestinal mucosa.

It occurs in those who are genetically predisposed,

and it resolves, the damage resolves, with

exclusion of dietary gluten.

Here, on the left, is a normal intestine

with a normal villus structure; and on the right,

fully evolved celiac disease with complete

destruction.

The villi are gone, not only are they gone

but this entire intestinal mucosa is greatly

thickened and filled with inflammatory cells. This

is where the primary site of injury occurs in

celiac disease.

I didn't mention it, but it is a permanent

condition. While it will heal most of the time

with exclusion of gluten, the intolerance to gluten

is permanent and will recur when the individual is

reexposed to gluten.

(Slide.)

DR. MURRAY: Now, what causes celiac

disease?

(Slide.)

DR. MURRAY: We know there are two major

components to this disease: the first is the

genetic background of predisposition.

Much of that predisposition revolves in

the HLA type, which is part of our human leucocyte

antigen-recognition system. It is how we determine

self- and non-self and generate an immune response

as appropriate and its interaction with

environmental factors, primarily the environmental

factor of gluten.

These two conspire together to produce an

immune response that becomes out of control

resulting in inflammation, which we just showed to

you, that occurs primarily in the proximal small

intestine and then subsequently the consequences of

this inflammation leading to malabsorption and

symptoms.

(Slide.)

DR. MURRAY: What do we know about the

genetics of the disease? For many years, we know

there is a strong, familial predisposition to the

disease.

If you are unlucky enough to be a

monozygous twin of somebody affected with celiac

disease, your concordance rate is 80 percent. It

is not 100 percent, but it is about 80 percent. If

you are a sibling of a celiac, your chance of

having it is 10 percent. If you are a child of,

about 5 to 10 percent.

There is a very strong association with

certain HLA molecules. These are Class II MHC

molecules but particularly two types. First, DQ2

is the predominant type that is required for celiac

disease, and in some populations DQ2 is also an

enabling type.

These genes, however, while they are

essentially required for the disease, are not

sufficient alone to the development of the disease.

Probably 30 to 40 percent of the Caucasian

population carry one or both of these molecules,

but most of them don't get celiac disease. There

are other HLA genes that are likely involved,

though they have not been well elucidated and

certainly not confirmed in many populations.

There are other chromosomal disorders --

Down's syndrome, Turner's syndrome, and Williams

syndrome -- that are associated with a greatly

increased risk of developing celiac disease for

reasons that are not entirely clear, but probably

are associated with the increase risk of disease in

those chromosomal disorders.

(Slide.)

DR. MURRAY: Looking at the primary

environmental trigger for the disease -- that is,

gluten -- it is basically the storage proteins that

come from these particular cultivated grasses:

wheat, barley, rye, and other similar grains from

within those families. Other grasses -- for

example, rice, items such as corn, sorghum, millet,

and probably not even oats -- are not involved in

triggering the disease.

(Slide.)

DR. MURRAY: It is the storage proteins

from the endosperm compartment of the wheat kernel

particularly, and those are gliadins oar glutenins

that are thought to contain the antigenic moieties

that trigger the disease.

(Slide.)

DR. MURRAY: What is it about these wheat

proteins? Well, if you take wheat, as an example

of the others, these storage proteins are uniquely

high in certain amino acids, especially glutamines

and prolines.

Over 30 percent of the amino acids in

gliadin are glutamines. The glutamines, of course,

can be cross-linked to give the grain its

resiliency. Really the cooking ability, the

ability to use wheat as such an effective way of

making things that stick together like bread, for

example, and maintain their shape, is largely a

property of these particular combinations of amino

acids.

The proline sequences that contain or

proline residues contained within the wheat

proteins also appear to form helices, and these

helices are resistant to digestion within the

mammalian gut.

(Slide.)

DR. MURRAY: This may be a key factor in

what results in the likelihood of these peptides

basically being maintained and becoming, then,

still available for the immune system to recognize

in a patient with celiac disease.

Now, gliadin molecules are presented by

these HLA types to T-cells in the intestine, and

T-cells that are specifically primed to respond to

gluten. There are certain gliadin molecules that

have a higher affinity than others for these

Class II molecules and then the T-cell receptor.

These peptides may be processed or altered

within the gut, perhaps, to make them more

antigenic. They may not start out very antigenic,

but then they undergo some change within the gut

that may make them more antigenic.

It turns out that some of these peptides

that are particular immunodominant, these are the

ones that are most likely to produce an immune

response, that those immunodominant peptides may be

digestion resistant because they contain those

proline sequences that perform helix, making them

relatively poorly digestible by peptidases within

the gut.

(Slide.)

DR. MURRAY: Now, it turns out that there

is a contribution to this antigenic nature from

within the intestine itself, and this may well be

because of this enzyme tissue transglutaminase.

This is an enzyme that is present within

the gut mucosa. It is released by cells,

especially fibroblasts when they become inflamed,

and it cross-links cystine residues.

It turns out that it will also act on

gliadin by deamidating some of those glutamine

residues, some specific glutamine residues, to

glutamates, making it more antigenic by deamidating

that gliadin peptide and making it fit more

perfectly or with a tighter affinity into the

binding groove of the DQ2-HLA molecule, and, hence,

producing a more vigorous immune response within

the gut.

(Slide.)

DR. MURRAY: This is a schema, a

relatively simplified schema, of what I have just

talked about. We start with wheat. You look at

particularly the ethanol-soluble fraction, and

gliadin is probably broken up into smaller

peptides, but still of a sufficient size to produce

an immune response.

It is taken up across the epithelium

presented by antigen presenting cells to the

T-cells. These are T-cells that will specifically

respond to gluten then producing two types of

responses: a cellular response, characterized by

lymphocytes producing interferon gamma and possibly

other cytokines. It is probably the cellular

response that leads to the "inflammatory cascade"

that produces the damaged epithelium characteristic

of celiac disease.

It also produces help to the B-cell side,

to produce plasma cells that produce antibodies.

These antibodies are directed both against the

exogenous antigen gliadin and also antibodies

against tissue transglutaminase or what was known

as an the endomysial antibody. It is not known

what the actual pathogenic role of this is, but it

is a very useful serologic or blood marker for the

disease.

I mentioned about the antigen getting

changed by tissue transglutaminase. This is a

little cartoon which shows the peptide derived from

gluten. If you change one specific glutamine to a

glutamic acid, which could be done by tissue

transglutaminase, this then binds much more

tightly. This is the HLA molecule here on the

surface of the antigen presenting cell, and it fits

more perfectly into the T-cell receptor, producing

a more potent T-cell response.

(Slide.)

DR. MURRAY: Now, there are other things

that happen in the setting of celiac disease, and I

am really touching just on the surface of many of

these, but there are other things that result in

this inflammation that damage the lining of the

intestine.

For example, there are metallic proteases

that damage the structural elements that maintain

the structure that maintain villus structure.

There is endothelial injury that occurs affecting

the blood vessels in the villus. There are

antibodies, autoantibodies, that are produced that

affect actin that are involved in the site

maintaining the structure of the entrocyte itself.

(Slide.)

DR. MURRAY: Recently, there has been work

suggesting that there is a molecule called

"zonulin" that may be released in the setting of

celiac disease.

This is important because it opens up

tight junctions between entrocytes which may allow

even more ready access of the antigen, the foreign

antigens, between the cells into lamina propria

where antigen-presenting cells can then present

those peptides to the gluten-responsive T-cells,

further accelerating the disease.

(Slide.)

DR. MURRAY: Now, I pointed out that many

people, 30 percent or more of the Caucasian

population, carry DQ2 or DQ8. Virtually, the

entire population are exposed to gluten, but most

people don't get the disease.

There must be triggers that produce the

disease. There is evidence that suggests that

gluten in the infant diet, specifically the age of

introduction of gluten into the infant's diet, may

be important in triggering or at least producing

autoantibody markers suggestive of celiac disease

early in life.

It is not clear, however, if that changes,

whether you delay introduction or not whether that

changes, the lifetime risk of celiac disease, but

it certainly seems to be important in triggering or

producing evidence in childhood at least of celiac

disease immune markers.

The amount of gluten in the child's diet

may be important. There are other events such as

pregnancy, infection, or surgeries that may bring

previously asymptomatic celiac disease to clinical

presentation.

(Slide.)

DR. MURRAY: One could speculate, and I

think this is based on some data, putting data

together, that one's risk for celiac disease starts

with your HLA type. Only those who carry HLA types

are at risk. You, then, are exposed to gluten.

Perhaps the timing of exposure is important,

developing in some individuals a sensitivity to

gluten.

Then, with the interaction of other

factors such as other genes other than HLA, other

things that may predispose one to autoimmunity

including gender and other events that may occur --

gastroenteritis, aging, postsurgical or postpartum

changes in the immune system that may occur -- may

lead to a loss of tolerance, inflammation, and

subsequent malabsorption.

(Slide.

DR. MURRAY: Don Kasarda, who is here once

used the term or suggested that celiac disease was

a collision between our evolution of our immune

system and our ability to recognize self and

non-self through the HLA system and our cultivation

of wheat and these other grasses. This collision

occurs in the intestine.

(Slide.)

DR. MURRAY: Now, when this collision

occurs and results in damage, how does it present?

And who gets the disease?

(Slide.)

DR. MURRAY: Well, this is classic celiac

disease, and this is the way that I certainly

learned about celiac disease. A severe

malnourished child with evidence of malnutrition

often associated with the large, swollen abdomen

but great muscle, terrific muscle, wasting and

protein-calorie malnutrition with symptoms that

would occur sometime after the onset of gluten

introduction into the diet, sometime between the

age of six months and seven years of age: with

failure to thrive; abdominal distention; anorexia;

diarrhea; steatorrhea, that is the passage of

malabsorptive stools laden with fat; anemia; growth

failure; and vitamin deficiencies. That was really

the picture that we had of celiac disease 30 years

ago.

(Slide.)

DR. MURRAY: However, we now see celiac

disease in adulthood. In fact, celiac disease can

present at any age. Symptoms can include things

such as abdominal pain, even upper-GI symptoms:

heartburn, nausea, vomiting, anemia, fatigue.

There are of course patients who have

symptoms of malabsorption, though not necessarily

the classic, fully evolved malabsorptive picture.

Steatorrhea as a presenting symptom is relatively

rare, even patients may have constipation.

(Slide.)

DR. MURRAY: It can mimic other disorders

such as lactose intolerance. Indeed, lactose

intolerance may be secondary to the damage caused

by celiac disease. It may mimic the symptoms of

irritable bowel syndrome or symptoms of

inflammatory bowel disease.

(Slide.)

DR. MURRAY: There are specific

deficiencies that can occur in celiac disease,

especially the fat-soluble vitamins -- D, E, A,

and K -- with their resultant syndromes from

deficiencies.

Iron deficiency is especially common in

celiac disease because iron is absorbed in the

proximal small intestine; folate deficiency, again,

because it is absorbed in the proximal small

intestine; and, interestingly, B12 deficiency may

be relatively common in celiac disease by a variety

of mechanisms. Other trace elements -- zinc, B6,

selenium, and others -- may also be deficient in

celiac disease.

While in the past we would look for

combinations of these, often a patient would

present with many of these deficiencies at the time

of diagnosis, now it is relatively uncommon to see

the entire spectrum of deficiencies. Indeed, you

usually see one or two deficiencies that are

clinically evident, and the others may not even be

present.

(Slide.)

DR. MURRAY: How about non-intestinal? I

have mentioned that the major site of injury is in

the gut, but there are patients who will present

with non-intestinal presentations which can involve

into things such as the musculoskeletal system,

joint pains and osteoporosis or osteomalacia;

infertility or reproductive issues, delayed

puberty, spontaneous recurrent abortions have been

described; hematologic, which is predominantly

anemia; hyposplenism is an unusual consequence but

can present; and then dentition, enamel defects,

can be a presenting feature.

(Slide.)

DR. MURRAY: To focus on iron deficiency

anemia, which is probably one of the most common

reasons that I see celiac disease, about 5 to 8

percent of adults who present with unexplained iron

deficiency in some studies have celiac disease.

It is especially common in those who are

resistant to the use of oral iron. If you look at

individuals who are coming to gastroenterologists

for endoscopy, it may be 5 to 15 percent of those

patients, depending on the study, who may have

celiac disease if biopsies are taken from the small

intestine.

However, many of those patients are even

missed because the biopsies are still not taken

during routine endoscopy in patients who have got

anemia in about a third to a half of patients.

(Slide.)

DR. MURRAY: Osteomalacia or bone disease,

this is an example of severe disease with

pseudofractures in the pelvis of an individual,

whose only presentation was osteomalacia with no GI

symptoms, caused by celiac disease.

Other non-intestinal presentations include

neurologic or even neuropsychiatric syndromes such

as neuropathy, ataxia, seizures, cognitive decline,

or dementias; fibromyalgia-like syndromes or

chronic fatigue syndrome-like presentations;

individuals with skin and mucous membranes, there

is a specific rash associated with celiac disease,

a recurrent aphthous ulceration of the mouth; the

dental enamel defects we mentioned.

(Slide.)

DR. MURRAY: And, then, dermatitis

herpetiformis was specifically mentioned, because

this very blistering, extremely itchy skin rash

that affects the extensor surfaces is a direct

manifestation of intestinal gluten sensitivity.

(Slide.)

DR. MURRAY: Now, what about other

associated conditions? Celiac disease is

associated with other autoimmune conditions. It

may be seen in 3 to 7 percent of Type I diabetics,

individuals with thyroid disease, individuals with

inflammatory arthritis, primary biliary cirrhosis,

as examples of others; and then the congenital

disorders, especially those associated with

chromosomal abnormalities and also selective IgA

deficiency. If you look at relatives of celiacs,

it is anywhere between 5 to 20 percent, depending

on how one is related to someone with celiac

disease.

(Slide.)

DR. MURRAY: Now, beyond the symptomatic

celiac disease, there are also individuals who have

no symptoms, who already have fully evolved damage

within their intestine, and there may be no symptom

or there may not be occurring in someone with an

associated disease.

This is frequent to find this in first-

and second-degree relatives of patients with celiac

disease.

(Slide.)

DR. MURRAY: There is also what is termed

"latent" -- well, whether it is latent celiac

disease or latent gluten sensitivity -- individuals

who have a positive serologic response but have a

negative small-bowel biopsy. Some of those

patients will go on to develop the full-blown

disease, if followed, on a normal diet.

(Slide.)

DR. MURRAY: What about the epidemiology?

To summarize, while it was first identified in

Europe, it occurs essentially in all populations,

which could be termed Caucasian. Its prevalence is

probably somewhere between 1 in 90 to 1 in 300;

however, the diagnosis rate is much lower, which

would suggest a prevalence of about 1 in 2,000, if

you just look at the diagnosed cases.

It is one of the most frequent genetically

based diseases. If you look at other countries --

Latin America, or other areas; Africa, especially

North Africa; if you look at Asian countries --

there is celiac disease present in those. The

worldwide average prevalence is somewhere very

close to 1 percent.

(Slide.)

DR. MURRAY: I'm coming a little closer to

home. This is the data from Olmsted County, that

we published a couple of years ago, which looked at

the new case identification or the incidence rate.

The solid yellow line is the new cases per

100,000 in the population, which is essentially

quite low and stable over many decades until the

1990s and into 2000 to 2001, showing a greatly

increased rate of detection of celiac disease.

If we looked at who were being diagnosed,

this is the age of diagnosis by age category. This

is the incidence per 100,000 of people in that age

category in the community. You can see that the

new cases being diagnosed are predominantly people

between the age of 45 and 64.

The solid line indicates females; so,

females are diagnosed at a rate about twice that of

males of all ages.

There are a significant portion of

individuals, almost a third, who were diagnosed for

the first time over the age of 65.

(Slide.)

DR. MURRAY: One can term or consider

celiac disease like an iceberg. These are a series

of icebergs where the tip of the iceberg is the

part that has been detected, and the part

underwater is the part that can be found if one

screens the population. Of note, these are numbers

per thousand not percent.

Obviously, there are some places like

Ireland, for instance, which is quite a big iceberg

with a lot above water but also a lot below water.

Finland, which this iceberg is probably even more

out of the water as they have got a very active

program for finding celiac disease.

This is circa 1996. The U.S.A. iceberg is

still close to a very low level of actually being

diagnosed; but this number, the part underwater,

has actually grown to be something very close to

what you would find in Finland or Ireland,

especially when one looks at, at least what we know

about is really from a Caucasian population.

(Slide.)

DR. MURRAY: One miniature study, this is

one we simply looked at, Natrona County in Wyoming.

This is a very isolated community. Anybody who has

been to Wyoming, there is lots of nothing for miles

and miles.

We were able to study about 4,000

individuals from a health fair, generally healthy,

and found the numbers above just under 1 percent of

people who had serologic evidence for celiac

disease.

Only half of them had GI symptoms. Most

of them did not have other risk factors for celiac

disease, just two having family members with celiac

disease.

However, these are numbers that would

confirm basically the rest of the world's data that

suggests that the prevalence of celiac disease, if

you look for it, is probably slightly under

1 percent.

(Slide.)

DR. MURRAY: Now, how does one make the

diagnosis? It starts with suspicion. Serologic

tests may be very effective at detecting the

disease. The intestinal biopsies are regarded as

the gold standard. Then, one ultimately gets a

response to a gluten-free diet to confirm the

diagnosis.

(Slide.)

DR. MURRAY: The pathology, as we have

mentioned already, is a pathology of chronic

inflammation within the intestine with features

such as intraepithelial lymphocytes on the surface,

villus atrophy or the loss of the villus surface,

great crypt hyperplasia, and then characterized by

being a lamina propria filled with lymphocytes,

macrophages, plasma cells, and even eosinophils.

(Slide.)

DR. MURRAY: There is however a spectrum

of damage that occurs, typified here by the Marsh

classification. This is classic disease, but there

are also milder forms of the disease that may be

asymptomatic in most individuals.

(Slide.)

DR. MURRAY: Our algorithm for finding

celiac disease, if we have a high clinical

suspicion, an individual with malabsorption, we

biopsy those individuals. If we have an individual

who is at moderately increased risk, serology is

probably the most effective way of finding it.

Though, we yet do not depend on the serology alone

to detect this, there are other circumstances for

alternate; serologic testing may be necessary.

(Slide.)

DR. MURRAY: Now, what about treatment for

celiac disease, or, as one patient with celiac

disease called it, "playing gluten-free roulette"?

(Slide.)

DR. MURRAY: The nuts and bolts of a

gluten-free diet, basically one needs to avoid

foods that contain the offending grains: wheat,

barley, rye, and the wheatlike grains of spelt and

kamut.

I put down at the bottom that many corn or

rice commercial cereals do not appear to be

gluten-free because of their incorporation of

particularly barley extract in their flavor

ingredients.

(Slide.)

DR. MURRAY: Now, one of the issues and of

course the issue for today is, How much gluten is

too much? We will be hearing a lot more when you

hear prospective and retrospective data later this

morning on this. I am not going to dwell on it.

One thing from clinical appreciation is

that symptoms are not a good indicator of gluten

ingestion. Many patients can have significant

damage to their intestine, despite the absence of

symptoms when they ingest gluten.

Most patients diagnosed clinically with

celiac disease have never suspected that wheat or

gluten products are what are precipitating their

symptoms. They may have or are often likely to

have blamed other foods that they weren't able to

digest because of the damage.

Antibodies such as tissue transglutaminase

antibodies are really only positive of the

substantial gluten contaminating the diet. At

least in my practice if somebody admits to cheating

more than once a month they will like continue to

have injury in their gut. However, there is a high

degree of variability in the sensitivity to gluten

ingestion, at least clinically.

(Slide.)

DR. MURRAY: We will hear a little bit

about Codex Alimentarius draft standards. This,

however, is still a draft, I think a Stage IV

draft. I want to put this up really to demonstrate

that there is a variance between countries and what

one allows.

We will hear more from our colleagues from

Europe about some of the incorporation of rendered

gluten-free foods which use the gluten-containing

grains as a base and then remove the proteins from

them.

(Slide.)

DR. MURRAY: What about non-responsive

celiac disease? This is relatively common. By

far, the most common reason is inadvertent gluten

contamination of the diet and lymphocytic colitis,

pancreatic insufficiency, bacterial overgrowth, and

then only a few patients have true refractory

disease that no longer responds to exclusion of

gluten from the diet.

(Slide.)

DR. MURRAY: There are many potential

sources of contamination of the diet with gluten,

which include of course commercial cereals, eating

out, communion wafers, lipstick, airborne flour or

starch in certain work situations, so-called "soy"

sauces made from wheat, mislabeled or unlisted

ingredients have been an issue, and at least

allegedly some medications.

(Slide.)

DR. MURRAY: Some ingredients that people

are concerned about: seasonings and spice blends,

modified food starch, malt and malt extract,

modified hop extract or yeast-malts, sprout

extract, dextrins, caramel color. There are a

whole bunch of things that might be derived from

gluten-containing grains.

(Slide.)

DR. MURRAY: What about complications

associated with untreated celiac disease? We know

the mortality of symptomatic celiacs who do not

comply with the diet has doubled. The mortality of

celiac disease even when it is diagnosed is also

double, even following out for several years after

the diagnosis.

The predominant excess in death comes from

GI malignancies. There are also morbidity

consequences such osteoporosis or osteomalacia,

stunted growth, infertility, chronic ill-health --

all of which could be prevented by early detection

and treatment.

(Slide.)

DR. MURRAY: What are the dangers of

non-compliance? The increased mortality we

discussed, the osteoporosis. Children who were

diagnosed and then did not remain on a gluten-free

often have osteoporosis or diminished bone density

when they get to adulthood, lymphoma, other

cancers, and then the psychological effects of

non-compliance.

(Slide.)

DR. MURRAY: One of the most feared

complications of this are the T-cell lymphoma.

This is celiac disease on this side, and the T-cell

lymphoma on the other side is one of the more

feared complications with a very high mortality.

(Slide.)

DR. MURRAY: However, as I pointed out,

most celiac disease is probably not symptomatic, at

least when we look at a cross-section of the

population. We do not know whether those

identified by screening are less sick than

clinically diagnosed celiac disease.

We don't know the benefit or negative

effects of a gluten-free diet in those who are

found by screening alone. We don't know if they

are any more or less likely to comply with a

gluten-free diet. We don't know whether

intervening in those patients will actually affect

their ultimate mortality.

(Slide.)

DR. MURRAY: George Dennison Prentice

said, "What come call health, if purchased by

perpetual anxiety about diet, isn't much better

than tedious disease."

(Slide.)

DR. MURRAY: This comes to the future.

There is, I think, a promising future in celiac

disease, a variety of approaches, which I have

listed, that individuals and research groups are

looking at as alternates to the gluten-free diet,

though none of them are really even close to

clinical use.

(Slide.)

DR. MURRAY: This is one that has been

tested in patients using a lactobacillus digestion

strategy, trying to reduce the potential, harmful

effects of gluten.

In summary, I would like to suggest that

gluten or celiac disease is common. It has been

largely unrecognized until recently. There are

many challenges that face patients and their

physicians in the treatment.

The gluten-free diet is not simple. There

is widespread use of grain proteins in good, and

that makes it challenging for individuals with

celiac disease. Food ingredient source

identification is of great concern to patients.

Dietitians and those who counsel patients

with celiac disease, we are here because of

regulation or potential regulations. Defining

acceptable thresholds and verification of those may

be very important to patients with celiac disease.

(Slide.)

DR. MURRAY: I finish with an aside on

another food safety issue, an invitation to come to

Minnesota and enjoy Joe's and have worms at the

same time.

Thank you.

CHAIRMAN DURST: Thank you very much,

Dr. Murray.

QUESTION-AND-ANSWER SESSION

CHAIRMAN DURST: I would like to ask the

Committee if they have any questions or discussion,

and also to point out that Dr. Murray will not be

able to stay around for discussion this afternoon.

If there are questions, now is the time to ask

them.

Doug.

DR. HEIMBURGER: Thank you very much for

that presentation. It is very helpful. I do care

for some patients with celiac disease. As you

already mentioned, one of the big questions that

they have is, "What really are my risks if I play

with it a little bit? If I knowingly introduce a

little bit of gluten in my diet, how absolutely

obsessive do I need to be about it?"

With the mortality being primarily

associated with the risk of lymphoma, what is the

quality of the evidence that compliance with gluten

restriction is really correlated with the risk of

lymphoma?

DR. MURRAY: The evidence, the quality of

the evidence is largely observational, taking

cohorts and following them, and often their

self-assessment of their level of compliance. The

data is based on those cohorts. They are

predominantly referral cohorts.

The longest follow up comes from Britain.

In those, there is quite a clear increased risk not

just for lymphoma but other malignancies in those

who are considered not to be completely compliant

with the diet. I would say the level of evidence

while it is not a prospective study, in retrospect

in fact it appears to be reasonably strong.

DR. BRILEY: Margaret Briley. I would

like to know what you can tell us what might be the

reason for the increased detection of celiac

disease? Is the serological test the definitive

reason? What is going on with those? It seems to

be more prevalent, as you said, in our society.

DR. MURRAY: I think there are probably

two reasons, one is probably serologic detection

has made this an acceptable diagnosis to primary

care physicians, number one; and the second is

suspicion, that is, the awareness of celiac disease

as a possibility.

I do not believe, however, we can rule out

the possibility that celiac disease is actually

increasing in prevalence over time.

We don't know, but we know that there

certainly were substantial increases in other

inflammatory bowel diseases over the last 50 years,

which may not have been accounted for by detection

rates, for example.

I do agree with you it is probably the

sero-detection. It is a combination of those two

things, suspicion and ease of detection have made

the biggest difference. However, I would not rule

out the possibility of its actually increasing in

prevalence.

CHAIRMAN DURST: Soheila.

DR. MALEKI: Soheila Maleki. I was

wondering how long does a person that is

asymptomatic go before they find they are

symptomatic? How do they usually find out?

DR. MURRAY: First of all, we don't know

when it starts. There is one study I didn't talk

about from Denver that suggested that children, if

you follow children at genetic risk for celiac

disease based on HLA type, that they will convert,

sero convert, by about the age of seven, though

most of them are asymptomatic.

It reaches all but 1 percent of that

childhood cohort, the same prevalence you find if

you look at adults. The suggestion is that you

have developed that celiac autoimmunity probably by

the age of seven.

However, the age, the median age, of

diagnosis is 45 years of age. It is likely that

those individuals have some clinical disease for a

long time before they present.

In our population, the most common reason

for presentation is postmenopausal anemia. Women

who have anemia sometimes go a long time,

especially when they are menstruating. The doctor

says that menstruating is an excuse for the anemia.

Now that they are menopausal and are no longer

menstruating they no longer have an excuse for

their anemia, and they are referred for evaluation

for the cause of anemia.

Iron deficiency anemia at least is one of

the most common, but there are other things. For

example, the development of chronic GI symptoms.

Largely, as the earlier questioner said, probably

awareness of the possibility of this disease, with

primary care doctors using sero-diagnosis to find

celiac disease.

There a lot of atypical symptoms. There

are reservoirs of celiac disease, the Type I

diabetics, the family members of celiacs, those

with chromosomal disorders. As the doctors looking

after those people become more aware of celiac

disease, they are testing their patients more.

DR. BRILEY: Could you speak a little bit more about

the evidence that you have in regard to physicians

using the serological test? Is it a pretty common

thing to do it at the very beginning of a patient's

coming in with GI problems, or is it more likely it

is down the road a while?

DR. MURRAY: If you look at the data on

patients’ presentation time, from presentation to

diagnosis of celiac disease, it is somewhere

between 8 and 11 years from the time they present

to their physician with a complaint to the time

their diagnosis is made.

I think that time period is now beginning

to shorten, thankfully. The serology is done close

to the end of that period, so it is often only when

the suspicion is generated. Partly that is because

it is not considered at the early differential

diagnosis of celiac disease.

It is also difficult. It is difficult

because the symptoms are not very specific.

Fatigue and some bowel disturbance and a little

anemia, that is not a rare syndrome or not a rare

collection of symptoms you will see in individuals

in our community. It is hard to know where you

look for celiac disease and when you look for it.

It is often a very delayed diagnosis.

DR. BRILEY: Is the serological test one

that is pretty accurate? Could one count on it if

you did it early?

DR. MURRAY: If you use the more modern

autoimmune tissue transglutaminase test, for

example, it is reasonably good. It is not perfect,

but it is quite efficient at detecting celiac

disease at that level, in that period. It is not

perfect, though, but it is a pretty good test.

DR. BRILEY: It sounds like we need to do

some education, then.

DR. HEIMBURGER: A follow up to that --

Doug Heimburger -- what is currently understood to

be the sensitivity specificity of anti-tissue

transglutaminase antibodies?

DR. MURRAY: Depending on the study, most

studies suggest that the sensitivity of tissue

transglutaminase for celiac disease is in the high

90 percent, so the high 90s.

The specificity is probably also in the

mid- to high 90s, so quite effective when you are

looking at a population you are suspicious of the

disease.

DR. BRILEY: One more question.

Margaret Briley. Do we have any data that shows

the celiac patient also maybe has been identified

with a lactose intolerance? Is there anything that

combines those two that you would know or does

that--?

DR. MURRAY: The combination is probably

the damage that is caused by celiac disease, the

damage to the entrocytes affects your disaccaridase

including lactase throughout the surface of the

small bowel, and, hence, you get a secondary

lactose intolerance.

In general, if you think of the genetic

basis or the ethnic groups that are involved,

celiac disease tends to hit those of European

extraction more so. I know that population will

tend to retain their lactase activity for longer.

If you look at Subsaharan Africans, for

example, we don't know actually what the prevalence

of celiac disease is in Subsaharan Africans, but

they are individuals who tend to lose their

lactase.

Genetically, they are probably separate, but they

come together because of secondary lactase

deficiency caused by the damage in celiac disease.

DR. NELSON: Thank you, Dr. Murray. That

really was a very interesting presentation. I

think the last time I studied this was 30 years

ago.

CHAIRMAN DURST: Identify yourself,

please.

DR. NELSON: Mark Nelson. I wanted to

touch base on the couple of slides where you talked

about how much gluten is too much and the proposed

Codex definition for gluten-free foods.

You had a question mark after the

threshold for damage being 20 to 100 milligrams per

day, and I guess that is similar to the naturally

gluten-free foods the Codex proposed.

Then, it goes on to talk about cheating is

greater than once per month. Also, is there an

issue of cumulative exposure, 20 milligrams per

day, or is the cheating an excursion of whole wheat

crackers, for example?

DR. MURRAY: Well, first of all, the

comment on cheating that is what the patients tell

me, those patients who will admit to gross

cheating, that is, eating a piece of bread or a

cookie or cake, which is what I regard as an

obvious source of gluten.

However, that may reflect some other

background, but a lack of detail of care, for

example, attention to detail in the rest of their

diet, maybe what they are not telling us. That

only applies to what they have told me.

The issue of actual threshold, I have a

question mark after that because we will hear a lot

more detail, science, about threshold testing for

thresholds of gluten contamination.

The intermittent contamination, once a

month obvious contamination. Something that is, to

some degree, under the patient's control with

appropriate education and exposure to information

then essentially it is under their control.

It is the low-level contamination on a

regular basis from sources they are not aware of,

and those are the patients that I see that make up

the majority of the patients I see who have

difficulty. They are coming to me because they are

trying to be gluten-free, but they are having

contamination of their diet on a daily basis,

probably a relatively small amount. We will hear a

lot more about the threshold, the actual testing of

the threshold using that type of low-level daily

contamination.

DR. MALEKI: Soheila Maleki. Is there an

adult onset or spontaneous development of celiac

disease, or does this have to come with a genetic

component and hereditary?

DR. MURRAY: It probably only occurs in

people who have that HLA or genetic type. You have

to have it. However, that is 30-plus percent of

the Caucasian population.

Can it start first in adulthood? We have

very little data on that. The only data is the

stuff I have mentioned, looking at children and

showing about 1 percent by the age of seven or

eight, positive by the age of seven or eight, the

same prevalence if you look at adulthood.

There are a few cases of what we call

"latent celiac disease," individuals who have got

antibodies with apparently normal small intestinal

biopsies as adults, then go on over a space of

years to develop full-blown celiac disease within

years of that initial identification of a positive

serology. Those are very rare cases that have been

found.

Of course, if you find somebody who

suspects they have got a problem, they ought to

change their diet anyway, and that changes

everything. There is very little data, I think, to

be sure of whether it first occurs, starts, in

adulthood.

If you look at the age of diagnosis, your

eighth or ninth decade can be the first time that

you are diagnosed with celiac disease. A lot of

those patients have suspicious symptoms going back

many years.

DR. BRITTAIN: Erica Brittain. Can you

quantify the level of damage? Is that only

possible with the biopsy, and is that very

invasive?

DR. MURRAY: You can -- well, if you take

biopsies, you can quantify the degree of injury. I

showed that slide which shows a spectrum of injury.

It tends to be variable, even within the same

individual. It started in the first part of the

small intestine and extends a variable distance

down the small intestine.

There has really only been one study,

which was done in the early 1960s, of taking

multiple biopsies down the intestine -- a handful

of courageous volunteers.

It is not clear that there is a

correlation between the extent of injury and the

severity of symptoms. In fact, we don't know how

to predict the occurrence of symptoms in patients

with celiac disease, so that is a "black box."

DR. BRITTAIN: You are saying there is

really no simple way to quantify the extent of

damage? There is nothing that would correlate with

these multiple biopsies?

DR. MURRAY: Some people have suggested

that the level of the antibodies, the tighter the

antibodies might correlate: The higher, the

tighter, maybe the more severe the injury to the

intestinal biopsies.

When we take a biopsy of the intestine, we

are sampling a tiny fraction of a percentage of

1 percent, a fraction of 1 percent, of the

intestinal lining. We have tried to look using

other imaging techniques to assess the extent of

injury. Using some of those techniques seems to be

very variable between individuals, and it doesn't

seem to predict their symptoms.

Really right now it is a yes or no issue:

yes, they have celiac disease; or, no, they do not.

It is very hard to measure the severity of the

disease.

We can look at the severity of

consequences: have they developed osteoporosis,

what their bone density is, have they lost a lot of

weight, whether they have severe malabsorption

based on fat malabsorption in their stool. We have

got other measures to look at the consequences or

impact of the disease, those we can assess.

DR. BRITTAIN: Do you think that it would

correlate with the intestinal damage? Not

necessarily?

DR. MURRAY: We have tried. I would say

that the data is not very good on that. People

with very mild injury may be more likely to be

asymptomatic, but the data is not sound.

CHAIRMAN DURST: Suzanne.

DR. MALEKI: Suzanne Teuber. I had a

question about the neurologic presentations. I

have read some on screening of pediatric

populations, but with adults how often does it

present as dementia without it being diagnosed as a

GI problem? Is this something we should be adding

to dementia screening?

DR. MURRAY: It is probably relatively

rare. In fact, I think there has only been one

good study. Maybe Dr. Collin, who is here as a

speaker later, has done a study and has looked at

and reported on that.

We occasionally see cognitive decline at

the time of diagnosis, but there is really no good

epidemiologic study to address that. Some of the

other presentations, peripheral neuropathy, for

example, or ataxia, there is more data on it. Many

neurologists are beginning to include celiac

disease as part of their differential diagnosis for

those syndromes. Good epidemiology data is

relatively small, very little data.

CHAIRMAN DURST: Ciaran.

DR. KELLY: Ciaran Kelly. This is

actually more clarification than a question. Dr.

Murray is quite correct that there isn't a measure

of either severity of intestine abnormality or even

height of antibody levels that reliably reflects

the degree of injury or correlates closely with

symptomatology.

However, with treatment one can use a

decline in antibody levels as a crude indicator of

at least reduced exposure to gross amounts of

gluten. It is not a very sensitive indicator, but

it is useful. Of course, with repeat biopsy, if

the histology has revered to normal, that of course

can be used. However, the less invasive test of

following antibody levels is used clinically to

follow response.

DR. MURRAY: Quite right. I think you

will agree, Joe.

DR. KELLY: If the antibody levels aren't

dropping, that is used as an indication that the

patient is successfully on a gluten-free diet.

DR. BRILEY: Margaret Briley. Can you

give us any idea of any behavior data that you may

have received from your patients regarding their

willingness to try foods that are not gluten-free

labeled?

DR. MURRAY: Oh, well, there are many

different attitudes among patients with regard to

what they want to eat or what they are afraid to

eat.

I advise my patients to be prudent, that

they try to select things based on identification

of ingredients, source ingredients, not containing

things contained from gluten, the use of substitute

grains that are gluten-free.

Many patients are quite willing to do that

on their own. Many of them use support group

information where maybe a group has cooperatively

contacted manufacturers who in good faith provide

information on their source ingredients.

There are some patients who are entirely

paranoid about it, and want to obtain a kit to test

the food. I don't know that we've got a very

effective kit yet for testing food for gluten

contamination. There are many different attitudes.

Fear is a major concern among my patients.

I mean, fear of even the slightest potential, not

even actual but potential contamination. This can

verge on, "Do we avoid taking prescription

medications for things like hypertension?"

resulting in life-threatening changes to their

medication regimens because of fear of

contamination.

I would say fear is a major part or a

major influence on the quality of life. We will

hear more I think shortly on the impact of a

gluten-free diet on patients' lifestyles a little

later. Certainly that does affect a substantial

portion of my patients.

Patients go through a substantial grief

reaction and feel socially isolated because of

their difficulty of interacting with society,

because so much of our society activities or social

activities revolve around food. There is that

safety sense of insecurity, which I think pervades

or affects many patients with celiac disease.

DR. BRILEY: Thank you.

CHAIRMAN DURST: Soheila.

DR. MALEKI: Well, I just want to know if

there are any coordinated studies for a

determination of thresholds? I know you mentioned

the level of PPMs. Do you know of any studies?

DR. MURRAY: There are and you will hear

about them. There are both retrospective and

prospective studies, and you will be hearing some

data on those later this morning.

CHAIRMAN DURST: I have one question --

Dick Durst -- on the biopsy and histological

studies to see the morphology, morphological

changes, you showed from the shag rug. I am just

curious whether just one of these cameras you can

swallow would be able to detect those kind of

changes without having to go through a biopsy?

DR. MURRAY: Yes, you can detect them.

Nobody would suggest that it would replace the need

for biopsies to make the diagnosis. There is

really relatively little published data on it.

There has been a paper suggesting that you can see

those changes. With a magnified view, you can see

with a capsule.

Yes, I think you can identify those

changes in a lot of individuals with celiac

disease, maybe all of them. Although, I really

can't comment more on that, because it hasn't

really been studied in any great detail.

CHAIRMAN DURST: Erica.

DR. BRITTAIN: Erica Brittain. Do you

have any insight about the cumulative effect of

decades of low levels, very, very low levels of

gluten exposure? Certainly, we are going to have

to think about what chronic exposure could do when

we talk about the thresholds. Can you provide any

insight into that?

DR. MURRAY: Probably the best clinical

insight I can give are individuals who I see who

were diagnosed 20 years ago and have not come back

to medical attention in 20 years. I see those

patients maybe every week.

I would see somebody who is diagnosed 20

years ago, and they got instruction at that time

that allowed them to eat things like barley malt or

that people weren't really instructed about some of

those rice or corn cereals that may have contained

malt, for example.

Those patients come back with anemia,

chronic GI complaints, maybe not as severe as they

had initially, but they certainly have accumulated

some health morbidity over those 20 years. Some of

them will come back with frank lymphomas and will

end up with a mortal complication of their celiac

disease.

Yes, at least my clinical observation is

that I frequently see individuals with problems

that we get rid of, once they now move to a much

more strict gluten-free diet, by eliminating those

things -- largely, because in 20 years they didn't

go back and get more education and realize that you

had to exclude those minor ingredients. That is

one way of looking at the effects of decades'

accumulation of low-level contamination.

CHAIRMAN DURST: Dick Durst again. On

your slide that showed the various causes, the

different grains, and so on, I believe you

indicated oats was not one of the causes. Could

you expand on that?

DR. MURRAY: We will hear a little more, I

think, from Dr. Collin on that issue. While oats

had been thought to be one of the offending grains

in things done in the fifties and sixties, it turns

out from recent very well-done studies that it

doesn't appear to impair the healing of the

intestine in newly diagnosed celiac disease. It

doesn't seem to result in a significant worsening

of production of damage in patients who are already

diagnosed with celiac disease.

For the vast majority of celiacs, it is

probably safe in its native, pure form. However,

there are some sequences within oats that can

produce an immune response, at least in vitro, in

lymphocytes derived from a few celiacs.

It is not an absolute. There may be some

individuals with celiac disease that can respond to

oats, both in the laboratory test and possibly also

clinically there are a few.

There are probably a relatively small

minority of celiacs in which that occurs. A bigger

concern is the issue of contamination of oats with

other grains that are well recognized to cause

injury.

CHAIRMAN DURST: Marc.

DR. SILVERSTEIN: Marc Silverstein. I

would like to inquire about the potential

subsequent lifelong increased risk of GI cancers.

I presume that the risk is predominantly small

bowel, but I wonder if there is any increased risk

of colorectal cancer?

Then, what are your thoughts about whether

there is sufficient risk that patients with celiac

disease should be in some sort of surveillance

program for early detection of GI cancer?

DR. MURRAY: Clarifying the risk of

cancers, it is particularly visceral cancer but

also includes: esophageal cancer, non-Hodgkin's

lymphoma of any site not just the intestine, and

probably also B-cell lymphomas, as well as the

T-cell lymphomas, small-bowel carcinoma.

There is a greatly increased relative risk

of small-bowel carcinoma. Of course small-bowel

carcinomas is a very rare disease to begin with, so

the lifetime risk of dying of a small-bowel cancer,

even in a celiacs, is still relatively small. The

data on colon cancer is mixed. There is some that

suggests there is an association; and some, that

does not.

When you look at other causes of

mortality, even non-cancer causes of mortality such

as infections, neurologic disorders and chronic

lung infections, there are other excesses of

mortality that occur in patients with celiac

disease.

There are some reductions in cancer

mortality. It appears, at least there is a

suggestion, that breast cancer may be less common

in celiac disease than non-celiac disease. There

were a couple of suggestions that lung cancer might

be less common in celiac disease than in non-celiac

disease.

Now, whether there is some competing issue

like smoking may be less common in celiac disease

than non-celiac disease, so there may be some other

competing issues that are involved. Body size may

make a difference. It may be another confounding

issue that confounds or is a competing risk for

malignancies.

While small-bowel cancers and lymphomas

are the two that have the greatest relative risk,

it is a small, absolute risk because of the

relative rarity of those cancers.

CHAIRMAN DURST: Do we have any further

questions for Dr. Murray?

(No verbal response.)

CHAIRMAN DURST: Thank you very much.

Our next speaker is Cynthia Kupper, who is

the executive director of the Gluten Intolerance

Group of North America who will present on patient

perspectives on celiac disease.

PATIENT PERSPECTIVES ON CELIAC DISEASE

MS. KUPPER: Good morning. I am a

dietician, not a doctor. I appreciate the honorary

doctor status.

My job here today is to give a face for a

person with celiac disease. I have been tasked

with letting you know who they are, letting you

know how they get their information and education,

and then also providing you with some information

about labeling that they have.

(Slide.)

MS. KUPPER: First of all, living with

celiac disease is very difficult. It is a chronic,

lifelong condition, as you have heard, and people

find this to impact greatly their quality of life.

Forty-four percent of the patients in a

Canadian study say that the diet is very difficult

to follow. In fact, there are some studies that

suggest that the compliance with the diet can be as

low as 50 percent in teenagers but probably ranges

around 70 percent compliance.

Eighty-four percent of these patients in

Canada suggested they have a difficult time

determining what is gluten-free and what is not.

They don't travel, and they don't eat out. It

impacts their family life and their career.

If you have celiac disease and are an

X-ray technician, oftentimes you change careers

because sometimes the X-ray slides are dusted with

flour. Chronic exposure could impact your quality

of life.

(Slide.)

MS. KUPPER: I did a study online of 620

patients a few months ago. In response to that

study, 75 percent of them said that they can tell

the difference between a gluten reaction and other

intolerance or a food allergy.

When they discussed their reaction

symptoms, they ranged from anaphylaxis, which is

not a gluten reaction for celiac disease, to

delayed reactions which could impact any aspect of

their GI and other health systems, body systems.

The average time to reaction was somewhere

between four to eight hours, but some of them

complained of immediate, almost allergic type

responses, and many of them said that their

responses or the symptoms that they had would last

for several days.

Keep in mind, there is no medication we

can give them to make this go away, so they just

have to let it work its course. This is really

disturbing to me.

(Slide.)

MS. KUPPER: As a dietitian, patients do

not rely on medical communities and professionals

for their information. They rely primarily on

support groups.

Actually, the Internet should probably be

the first one, because they are Internet savvy.

They have been out there and they have gotten all

kinds of information before they ever see a

dietitian. Not only do they get information from

the Internet and the support groups, but there are

list serves and chat groups that they belong to.

These can be very useful tools for a

person with celiac disease. However, they also

provide some very frightening and unreliable

information that the patients will hold onto as if

it were gospel. Then, they work with self-help

books as well.

Unfortunately, doctors, and especially

dietitians, are seen as unreliable. It is sad for

me to say that as a dietitian my profession doesn't

get this disease. They also treat it like it is a

rare thing, and they don't know anything about it.

In the United States, I can tell you that there is

probably a handful of dietitians who would be

considered experts in celiac disease.

Doctors don't get much more respect,

primarily because it has taken so long for the

patient to get a diagnosis that the patient has

lost faith.

Then, they will go to research facilities

like the University of Maryland, Chicago, and

New York, or the Mayo Clinic. Lastly, they will go

to medical Web sites. The bulk of our information

is coming from potentially unreliable and

non-research-based sites.

(Slide.)

MS. KUPPER: The consumers perceive that

gluten exposure levels -- the question was asked to

me, "What do consumers believe about gluten

exposure? Are they concerned about the health

risks?" The answer is yes and no.

On the study that I did, it depended upon

their confidence of the labeling, and it depended

on whether they accepted testimony or accepted

research. There is a group of celiacs, as

Dr. Murray suggested, who really don't want to

listen to what research suggests.

As you move forward, with not only

establishing how you are going to determine the

threshold but what that threshold will be, you will

have a fight in the celiac community for a lack of

education and understanding of research.

Consumers oftentimes also have an

inability to correctly interpret research findings.

These are people who have just enough medical

knowledge to be dangerous, so they don't have a

full understanding of the terminology they are

talking about.

There is this constant perpetuation of

misinformation. I don't know how many times when

we try to bury something that is inaccurate it gets

dug up.

(Slide.)

MS. KUPPER: There are varying levels of

gluten sensitivity, as you heard, too. There is

the perception that gluten is poison. Not unlike

the allergy people that we heard from yesterday,

this is a huge issue to the celiac consumer.

They believe most of the time that when

their gut hurts it is from gluten not from

something else. Consequently, we are trying to

help the patient understand that not everything

that makes their gut hurt is gluten.

As Dr. Murray said, there is a huge fear

reaction. If I had to put a psychological label to

a group or at least a portion of the celiac

community, they are filled with fear and a little

bit paranoid about what they can and can't do.

How do we define "gluten-free" in the

U.S.? This is a really interesting question. Of

the consumers, only 19 percent realize that there

is no definition right now for gluten-free in the

U.S. Many of them define that the true definition

is zero. This is a problem -- a lot don't know.

(Slide.)

MS. KUPPER: When I ask the question, "Do

you trust gluten-free labeling?" It was

interesting, too, because most of the people say

they do trust it. However, when you ask them if

they ever had a reaction to a product labeled

gluten-free, you can see that up to 50 percent

suggested that they might have had a reaction to a

gluten-free product.

When I talk to manufacturers that

manufacture only gluten-free products and ask them,

"Do you test, and what do you test to," many of

them are using older testing methods not the newer

testing methods, the monoclonal tests that we

talked about yesterday.

Some of them tested 200 parts per million,

some of them tested 20 parts per million, some of

them tested no detectible. For the gluten-free

consumer today, the label "gluten-free" really has

no meaning.

(Slide.)

MS. KUPPER: Again, the gluten-free

consumer is compulsive about their medical needs.

This is their only treatment. It is often referred

to as our drug of choice. There is nothing else we

can do, except to follow a strict gluten-free diet.

They have very limited trust in the

manufacturing industry. They believe that labels

that say "may contain" and different things like

that need to be distrusted. When they call the

manufacturers, they are not quite sure that they

are getting the right answer all the time.

Also, they have a limited understanding of

what good manufacturing practices really mean, so

they are always questioning what the manufacturer

will say. Yet, at the same time they want

accountability and they want reliability.

They may translate information to the

extreme. Let me give you an example. A few months

ago on one of the list serves, someone put out a

message about bottled water being gluten-free.

That got taken in a week's time to the

point where consumers were calling asking why water

had gluten in it, and how dare the food industry do

that to them. The reality is it never did.

A company, out of the graciousness of

their heart, put it on a list of gluten-free

products, and from that the consumer decided that

every other bottled water had gluten in it. This

is the extreme that the consumer can go to. Again,

they don't find descriptive labeling helpful at

all.

The changes that can occur in ingredients

in manufacturing processes make it difficult for

this consumer group to know what they can have.

The term "modified food starch" usually means

cornstarch, modified cornstarch, in this country.

However, if the manufacturer determines

that wheat starch is cheaper in the fall and they

switch and the consumer has determined that this

product is gluten-free, now they are in trouble if

they don't recheck.

When you talk to the food industry, you

will find that their calls have dramatically

increased over the last 2 to 5 years of consumers

calling in, and 90 percent of the questions do not

have to do with other allergens but have to do with

gluten.

(Slide.)

MS. KUPPER: When the consumer asks the

question about gluten, the problem is that they are

asking the wrong question. The consumers believe

that if they don't have effective labeling how can

anybody possibly know that they are going to be

able to be healthy and protect themselves.

They want to know that if you call a

company they are really giving you the right

answer, and they are just never confident about

that. Oftentimes, when the company answers too

quickly, they get suspicious.

Oh, I've had that experience. I will call

on a product and I'll say, "I need to know the

source of the modified food starch."

"Oh, you're talking about gluten?"

"Yes. Tell me the source of your modified

food starch. Let me make that decision about

whether I'm talking about gluten." That makes a

consumer suspicious.

Finally, you know, if a person eats a

gluten-free food and they get sick, whether it is

related to gluten ingestion or not, they have

determined that they can't trust that company any

longer.

Again, these list serves and chat groups,

I have seen them take small companies out of

business because of the spreading of rumors --

which are probably unfounded.

(Slide.)

MS. KUPPER: In closing thoughts, I really

encourage that through this entire process related

to labeling thresholds, that we be talking a common

language.

Let me use the example of threshold.

Yesterday, as I listened to Anne Munoz talk about

thresholds for allergens, I realized that we have

three different definitions of thresholds -- or

tolerance, excuse me. I want to use the word

tolerance.

The consumer says, "Tolerance is zero."

What that means is they think there should be zero

gluten in their food.

The medical community says "zero

tolerance." For them that means, you should be on

a strict diet, and you should never cheat.

The manufacturing industry wants to know

where that is. Is it 20? Is it 200? They know it

is not zero.

We are not talking the same language. The

consumer needs to know that the manufacturer and

the industry or the legal ramifications around any

labeling are all using common terminology in a

language they can understand.

Education is a huge component. As much as

I am a supporter of this regulation and this law,

one of the things that is going to happen, as you

heard yesterday in discussions about soy lecithin

and other ingredients, it is going to become a bag

of worms. For the consumer, it is going to be very

confusing, and we need to have an education

component as part of the new labeling laws.

I encourage you, too, although you heard

it yesterday and you will probably hear it today,

too, we know that there is no testing kit available

that tests to zero. We know, as you will probably

hear later, that it is probably an impracticality

or unnecessary to even go there.

I implore that when you set a threshold

method and testing methods, when you set the

threshold level, that it be reasonable and

something that meets the health needs of the

consumer but also allows the industry to meet the

need.

(Slide.)

MS. KUPPER: As they found out in

Australia, when you set zero as the tolerance level

and as the magic number for food manufacturers, a

lot of gluten-free products that patients used no

longer can be labeled gluten-free. Now the

consumer is once again confused and outraged.

CHAIRMAN DURST: Thank you very much.

Do we have any questions for our speaker?

Yes, Jeff?

DR. BARACH: Hi. Thank you. Jeff Barach

with Food Products Association. If I interpret

what you said correctly, you were talking about the

consumer really doesn't find descriptive labeling

very helpful in the case of gluten-free.

I assume then the consumer would go to the

ingredient list or the 800 numbers or their

internal chat groups to find out whether the

product really is gluten-free or not. Am I

interpreting that right? Your constituency does

not want gluten-free labeling?

MS. KUPPER: I would say that is probably

right, that is the message I got from the survey.

In fact, they found that labels that say "may

contain" or "processed in a plant with" really is

frightening to them. They will look at a product

like that, and they will simply avoid it.

They do go to chat rooms and there are

lists of gluten-free products. However, when you

look at those lists and you ask how they were

developed, there are no standards for developing

those lists.

DR. HEIMBURGER: Doug Heimburger, a follow

up to that. That is not the same, is it, as saying

"gluten-free"? Do they not want a label except it

is gluten-free with a consistent and clear

definition of that?

MS. KUPPER: They do want a label that

says gluten-free with a clear and consistent

definition.

DR. HEIMBURGER: Yes.

MS. KUPPER: I believe that gluten-free is

not is not going to mean zero; it can't mean zero.

CHAIRMAN DURST: Any further questions or

comments?

(No verbal response.)

CHAIRMAN DURST: Thank you very much.

CHAIRMAN DURST: Our next speaker is

Dr. Donald Kasarda, who is a consultant and retired

senior scientist from the Agricultural Research

Service of the USDA. He will make a presentation

on grains.

GRAINS

DR. KASARDA: Good morning everyone. I am

a research chemist retired from the U.S. Department

of Agriculture, although I still maintain a

relationship with my old lab in Albany, California,

as a collaborator.

Now, Dr. Murray covered a lot of the

things I am going to talk about. Maybe I will be

able to add a little bit more detail to some of

them, but he did an excellent job of talking about

some of the grain topics.

(Slide.)

DR. KASARDA: Immunology textbooks often

classify hypersensitivities into these four types.

Celiac disease is a delayed type hypersensitivity

that involves T-cells in the primary mechanism. It

falls into Type IV. Allergy is Type I and is

mediated by IgE antibodies.

Now, in the case of celiac disease, it is

often suggested that there is a Th1 mechanism

involved in which T-cells are presented with

gliadin peptides, and, ultimately produce

cytokines, inflammatory cytokines such as

interferon gammas as an example.

Now, in the case of allergy, however, the

same molecules that can induce the symptoms of

celiac disease are also capable of producing

allergies. We do have a certain amount of

confusion sometimes between immediate

hypersensitivities and the delayed-type, celiac

disease.

(Slide.)

DR. KASARDA: Now, this is the same

diagram that Dr. Murray showed. I want to talk

about primarily the endosperm, which is this white

part here (indicating) in the cutaway diagram.

The starchy endosperm is made up of about

75 percent starch, but it also contains about 7 to

17 percent protein, depending on the use of the

wheat. Most of this protein, about 75 percent of

it, is gluten protein.

The proteins are storage proteins. They

are used by the developing plant that comes from

the germ here. The germ is separated from the

outer layers and the endosperm during the milling

process after crushing and sieving.

The storage proteins are broken down upon

germination of the seed to produce a new plant.

The resulting amino acids and nitrogen are used in

the synthesis of new molecules needed by the

developing plant. Now, as I mentioned, about 75

percent of the storage protein is, in fact, gluten

protein.

(Slide.)

DR. KASARDA: This is a picture of flour

particles, a scanning electron micrograph. These

round, spherical structures are starch granules.

These (indicating) are A type, there are some

B types which are small here. The surrounding

rough-edged material is the gluten protein or

storage protein.

(Slide.)

DR. KASARDA: If you mix together flour

and water, as most of you have had the experience,

you can form a cohesive elastic dough. If you need

a dough under water, say, in a large container of

water or under a stream of water, you can wash out

the starch granules; they pop right out of the

matrix. You are left with a cohesive, elastic mask

consisting mainly of the storage or gluten

proteins.

(Slide.)

DR. KASARDA: Now, this is the traditional

cereal chemist definition of gluten. You cannot

carry out this process with rye and barley.

Therefore, to the traditional cereal chemist, there

is no gluten in rye and barley. However, the

celiac disease community has adopted the term

"gluten" for any protein that is toxic or harmful

to a celiac patient.

This terminology problem sometimes is

confusing when patients go to a company where they

might be dealing with a traditional cereal chemist,

and there is a certain amount of confusion as to

what is gluten. As I said, this is the traditional

definition, but it has been expanded to include

other grains that are harmful to celiac patients.

Now, from time to time, you will hear

about these fractions of gluten. Going way back,

at least over a hundred years, it has been

traditional to divide gluten into two, roughly,

equal fractions based on their solubility. This is

not an exact separation. No solubility fraction is

ever perfect.

Traditionally, it was alcohol-water

solution and sometimes we used detergent solutions.

We divide it up into the soluble fraction, which we

call "gliadin."

This is made up of monomeric proteins of

the prolamin class. The prolamin terminology comes

from Osbourne back around 1900. It is derived from

the fact that there are two major amino acids found

in the composition of these proteins. Proline and

glutamine, hence, prolamin.

By structure, we have three types: the

alpha type, gamma type, or omega types. Sometimes

people speak of the alpha/beta. I will talk about

that in a little as we go along.

Now, the insoluble fraction is called

"glutenin" by the cereal chemists. In rye and

barley, there is an equivalent fraction that we

called just generically "glutelin."

Now, this polymeric fraction consists of

prolamin subunits. Again, large amounts of proline

and glutamine in the composition of the proteins.

These subunits are linked together by disulfide

bonds into a higher level of polymer.

Of course, a protein is a polymer in

itself. It is divided into two main types the

low-molecular weight and the high-molecular weight

glutenin subunits.

(Slide.)

DR. KASARDA: This just is a table showing

the percentages in the various types of proteins.

For example, you have the sum of glutamine and

proline ranging from about 40 percent up to about

80 percent in some of the omega gliadins.

This is pretty unusual to have such a high

percentage of glutamine and proline. This is key

to the toxicity, because the toxic sequences

involve glutamine and proline and usually an

aromatic as well, either tyrosine or phenylalanine.

(Slide.)

DR. KASARDA: Now, the terminologies that

we use really go back to early electrophoretic

studies in the late sixties and early seventies.

Again, if we follow this diagram here, this is an

acid gel in which the proteins are separated by an

electric field in a polyacrylamide gel.

The terminology actually came from a sort

of free-boundary electrophoresis that was carried

out at our Northern Regional Research Center back

in the sixties. When they developed the

polyacrylamide gel electrophoresis, it was found

that the fractions fit with the mobility in the

electrophoretic gel.

You have the alpha, fastest moving; beta;

gamma; and omega. Structurally, the alphas and

betas are pretty similar. Some people will talk

about alpha/beta types. I just lump them together

as alpha types.

Now, the alpha type and gamma type are

about the same size. If you carry out SDS page or

polyacrylamide gel electrophoresis in detergent,

sodium dodecyl sulfate, which is a very good

dissociating agent for proteins.

Reduced or unreduced the gliadins give a

pattern somewhat similar to this. It is not quite

as good at resolving alpha, beta and gammas as you

find in the acid gels where aluminum lactate was

one of the favorite buffers.

If we go over to the glutenin fraction,

and these are the subunits linked together by

disulfide bonds, if you try to take a purified

glutenin fraction and run it into an acid gel or

into a detergent gel, mostly you've just got a

little bit of streaking around the origin because

the polymers are too large to migrate into the gel.

(Slide.)

DR. KASARDA: Upon reduction, however, you

begin to see this type of pattern here in which

there is a group of high-molecular-weight subunits

and a group of low-molecular-weight subunits. This

only occurs for the glutenin fraction when you

reduce the disulfide bond.

(Slide.)

DR. KASARDA: This is a two-dimensional

pattern, electrophoretic pattern, of the gluten

proteins. All you have to recognize is that each

spot here represents a separated protein. There

are quite a few different gluten proteins, and we

can count easily 50, 60, 70 spots in such a

pattern. Therefore, there are at least 50, 60, 70

gluten protein components.

We know from genomic studies that, in

fact, there are probably at least a hundred genes

coding for these proteins, and probably several

hundred genes coding for the proteins. The loci in

the genome that code for these proteins are spread

out over about nine different loci in the genome.

Now, as far as we know, all of these

gluten proteins are toxic in celiac disease. This

group here (indicating) are the omega gliadins, and

they seem to be particularly active.

However, all of the gluten proteins have

been tested by Paul Ciclitira's group and

Peter Howdle's group in the U.K., and they all

indicated by direct installation into the small

intestine that these proteins, all of these

different classes are toxic.

These omega gliadins are noted for being

strong allergen in exercise-induced anaphylaxis.

They are one of the really strong antigens involved

in that particular allergy.

(Slide.)

DR. KASARDA: This is a schematic diagram

that illustrates the fact that all of these

proteins are noted for a repetitive domain in which

certain amino acid sequences are repeated over and

over again.

They are somewhat degenerate, but we can

derive consensus sequences. These are glutenin

subunits, gamma-type gliadins. These red,

staplelike lines indicate intramolecular disulfide

bonds.

In a glutenin subunit, we also have free

cysteines which can link up to another molecule to

form these higher-level polymers. For example,

here is an alpha-gliadin -- I'm going to talk about

this a little bit more -- the end terminal, or the

first half of the molecule, is made up of these

repeating sequences.

The second half is not repetitive and

contains most of the disulfide bonds. Toxicity

seems pretty likely to be limited to the repeat

regions. These are the high-glutamine, the

high-proline regions.

Now, the omega-type gliadin seems to have

lost -- well, we are not entirely sure whether they

lost this type of domain or not, but in any case

they are made up almost entirely of repeating

sequences.

(Slide.)

DR. KASARDA: This is a hypothetical model

of the gluten polymer or glutenin in which the

subunits are joined by intermolecular disulfide

bonds, there are also these intramolecular

disulfide bonds, to form a higher-level polymer

that provides elasticity to a dough.

The gliadins and the glutenins are

cohesive with one another, but the gliadins

contribute more to the extensibility of the dough,

and the elasticity comes primarily from the

glutenin fraction.

(Slide.)

DR. KASARDA: Now, here I show some of the

types of sequences that you find in the repeats.

Now, they look pretty similar, a lot of glutamine,

which is represented by "Q"; a lot of proline

represented by "P"; and usually an aromatic

residue, either phenylalanine, "F," or tyrosine,

"Y."

Somewhere, and these are often degenerate.

They are not exactly according to the consensus

that I show here. Somehow along the line these

sequences have acquired toxicity in celiac disease.

(Slide.)

DR. KASARDA: Now, this is the complete

sequence of an alpha gliadin. This is the

end-terminal region up here (pointing). It starts

at one, and there are 263 amino acid residues.

Here, note the predominance of the blue Q's and the

red P's for the proline and glutamine residues.

This half of the molecule here is the

repeat region. There is also this interesting set

of glutamines, which really hasn't been studied in

celiac disease. It is probably not toxic, but, as

I say, there has been almost no study of this

polyglutamine stretch here.

Note also these vertical lines here which

I show. Those are sites that we have observed

where cleavages occur with gastric enzyme, pepsin

and pancreatic enzymes, trypsin and chymotripsin.

Now, most proteins would be broken down by

the digestive enzymes into single amino acids or

very small peptides: diatride, tetrapeptides that

are easily absorbed, which are probably not toxic.

(Slide.)

DR. KASARDA: In the case, as Dr. Murray

mentioned, because we have a lot of proline which

interferes with the breakdown by the proteolytic

enzymes, we can get some pretty large stretches.

This stretch here from 31 to 55 right here is

something that we have tested as toxic.

Other people have dealt with sequences

from this stretch and found them also to be at

given toxic. The fact that this gliadin and

glutenin proteins are difficult to digest by the

digestive enzymes allows these toxic stretches to

exist longer than you would find for other

proteins.

Now, this half of the molecule has a fair

amount of glutamine and proline, but as far as we

know toxicity does not reside in this C-terminal

half or sort of the end of the molecule. It is in

sort of the forward end of the molecule.

(Slide.)

DR. KASARDA: Now, this is what I would

call my string of beads model in which I have just

taken that sequence, 1 to 263, and shown it as

beads on a string.

Each bead represents a different amino

acid. I tried to assign the different amino acids

different code words to distinguish them. This is

the end terminal region of repeats. This

C-terminal where we have the disulfide bonds here.

Toxicity resides in this part here.

This sequence here from 31 to 43 was

synthesized by Mike Marsh in the U.K. first, and he

instilled the synthetic peptide directly into the

small intestine of several celiac patients and

found changes in the mucosa that were indicative of

celiac disease. So this does seem to be a toxic

sequence.

Here I show a computer molecular model of

what that sequence would look like in the

polyproline II left-handed helical confirmation

that Dr. Murray mentioned. We think that these

peptides do have a strong tendency to assume this

polyproline II confirmation.

Here, I show the sequence in three-letter

code as a string of beads model and here just a

single-letter code. I know most people are not

used to dealing with these codes. I apologize for

using them in some of the slides, but often I am

just trying to make a general point, and you don't

really have to follow the sequences according to

their exact correlations with the amino acids.

(Slide.)

DR. KASARDA: Now, this is a list of some

of the either toxic or immunoactive peptides that

have been described in the literature. This is

from Sean, et al, from Chaitan Khosla's lab at

Stanford. The 33-Mer appears to be a very active

sequence. I have indicated some sort of homology

100

here by the yellow boxes here.

All of these sequences, with the exception

of this one, have been found to be toxic by direct

installation into the small intestine or they have

been found to stimulate T-cells, T-cell clones,

derived from biopsies of celiac patients.

These are just some of the toxic

sequences. We don't know all of the toxic

sequences at this point. There are certainly

others to be found, so it is a pretty complicated

situation in trying to sort out exactly what it is

about the sequences that produces toxicity in

celiac disease.

(Slide.)

DR. KASARDA: Now I want to move on and

talk a little bit about the other grains. If we

start with the class flowering plants, which is one

of the major divisions or plants in terms of

taxonomy.

We go down to the major two subclasses,

monocotyledones plants and dicotyledones plants.

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We can follow the monocots down to the Gramineae or

the grass family.

Here, we have only wheat, rye and barley

that are toxic. Triticale is a cross between wheat

and rye, and so would be expected to be toxic.

Now, oats I have had to put in both

columns, and I will explain why. There are many

other grasses in which the grains do not have toxic

proteins as far as we know.

There are only two grains that have been

studied with modern methods and modern approaches

to understanding their relationship to celiac

disease, and that is wheat and oats.

These others have often been studied very

minimally including rye and barley, but rye and

barley do contain proteins that have sequences

quite close to those in wheat.

We assume that rye and barley are probably

toxic grains according to the early results of

Dicke in the Netherlands back around 1950, where

they considered rye, barley and oats as part of the

toxic group.

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Now about 15 years ago, I suggested that

if there are only a few grasses that contain the

toxic sequences, and they are closely related as

you will see, and then there are many other grasses

that do not contain the toxic sequences.

I suggested that if you get into the dicot

group -- the buckwheat, quinoa, amaranth, and these

other grains -- it would not be toxic, simply

because of their distant taxonomic relationship to

wheat.

I was a little bit apprehensive about

suggesting this, but over the past 15 years since I

suggested this, people have been eating these other

grains. As far as I know, there hasn't been any

serious indication that these do, in fact, have

toxicity for celiac patients.

There have been some very fine studies

from Finland and throughout the world, indicating

that oats were safe for celiac patients. But

towards the end of last year the Oslo, Norway,

group under Knut Lundin and Ludvig Sollid. They

have found -- well, I'm getting a little bit ahead

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of myself. Let's deal with this slide first.

(Slide.)

DR. KASARDA: If we take a subfamily,

festucoidiae, of the grass family and we look at

the tribal level, and I made this slide before the

results from Oslo were published. The hordeae --

which includes wheat, rye, and barley -- were one

tribe. I thought that oats were probably

non-toxic, so I put them in -- well, they belong in

a separate tribe. It was only this one tribe that

had the toxic sequences.

Now, it is pretty certain that the oats

are toxic to a few probably rare individuals, but

we don't really know how this works out. They

found three celiac patients who definitely reacted

to oats by the same mechanism that they reacted to

gliadin peptides. This, I think, was pretty well

demonstrated by the work from Norway.

Now, here I show the proteins that you

find in wheat, gamma-type gliadins and related

low-molecular-weight glutenin subunits, they are

found in rye, barley, not in oats. Alpha-type

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gliadins are found in wheat, but you don't find

them in rye and barley or in oats, and so on down

the line.

Now, the avenins are a small fraction of

the total proteins in oats. These make up only

about 10 percent of the protein. Most of the

protein is oat globulin, which as far as we know if

not harmful or toxic to celiac patients. There are

low-molecular weight proteins related to the

avenins in rye, barley, and wheat.

(Slide.)

DR. KASARDA: Now, this is another one of

those sequence slides, but let me just try to make

a few points here. This top sequence is a gliadin

sequence. It starts here (indicating) and runs

down to here.

The bottom sequence is an avenin, which

shows a lot of homology with the C-terminal half of

this gamma-gliadin molecule. This is also true for

the alpha gliadins. Where the amino acids are the

same, I have colored them in blue.

There is a lot of homology here in the

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C-terminal half, but that is not where the toxicity

lies. The Oslo group has shown that this

particular sequence, which I have underlined, does

have certain characteristics including glutamic

acid at key positions that are important, as Dr.

Murray pointed out, for binding to MHC proteins.

Consequently, most of the repeat region is

absent from the avenins. There is just this sort

of residual section here, which does have a lot of

glutamine and a lot of proline and some key

glutamic acid sequences or amino acids that seem to

be responsible in these few patients that have been

studied for the toxicity. This sequence is

certainly capable of stimulating T-cell clones from

these patients.

I think that the evidence is pretty good

that there are at least a few -- it seems as though

there are probably rare individuals who respond to

oats and probably most celiac patients do not

respond to oats.

This is a rather puzzling situation,

because I have always thought of the proteins as

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being pretty definitive for celiac disease, that

all celiac patients reacted to wheat and probably

to rye and barley, and that this was part of the

definition of celiac disease.

Now we have a situation here where it

appears that some patients react to oats and some

don't. This is some ongoing research that needs

some elucidation.

(Slide.)

DR. KASARDA: Just to jump back into the

classification, this is another subfamily,

panacoideae. Here we have maize and sorghum and

millet. We have actually done a little bit of

end-terminal sequencing on sorghum and millet

proteins, and they do seem fairly close to the maze

protein. This would explain to some degree why

they are not toxic in celiac disease. As far as we

know, it is still wheat, rye, and barley that are

harmful.

(Slide.)

DR. KASARDA: Now, this is my last slide.

The currently favored method for determination of

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gluten in food is the R5 monoclonal antibody ELISA

test developed by Mendez in Spain. This seems to

be a pretty good test; it is not perfect.

The antibody reacts to monomeric wheat,

rye and barley prolamins, but not the oat avenins,

and it reacts weakly or not at all with the

glutenin or glutelin, pretty good sensitivity,

recognizes these particular motifs more strongly,

although some others that are similar are

recognized weakly.

The Codex Committee on Methods agreed in

2004 to endorse temporarily the R5 ELISA for the

determination of gluten. Now, there are some

possible problems. There is the failure to detect

the glutenin proteins.

In some preliminary work from our

laboratory, for example, when we look at wheat

starch that is intended for use by celiac patients,

we find that the gliadins are pretty well washed

out, but we do find evidence of

high-molecular-weight glutenin subunits attached to

the starch surface. These would not be picked up

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by the R5 ELISA.

Then, there is the question about small

peptides from hydrolases. There has been some work

described using a competitive assay, which might

possibly solve the problem of the small peptides

from hydrolases.

Also, there is a certain amount of data

indicating differences in the results from

different labs on the same sample. On the whole,

it seems like a pretty good test that can be used.

As I say, it is not perfect, but it is a pretty

impressive test on the whole, and it may be as

close as we are going to get.

Although, I certainly can think of some

ideas for maybe improving it. At any case I think

I will end my talk here and I thank you very much

for your attention.

CHAIRMAN DURST: Thank you.

QUESTION AND ANSWER SESSION

CHAIRMAN DURST: Are there questions?

Okay. Margaret?

DR. BRILEY: Margaret Briley. Can you

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give us any idea of the use of this ELISA test by

industry in terms of the frequency and the

acceptance and willingness to do it? Do you have

any feel for that?

DR. KASARDA: Well, I think it is being

used, and Dr. Collin can comment on this,

extensively used in Europe and it is becoming used

in the U.S. Susan Hefle, who spoke yesterday, I

think that they have done a certain amount of work

using this test for industry.

My impression is that not much testing is

done in the U.S. Maybe Cynthia could comment on

that. I think it is developing, but we are quite a

bit behind the Europeans in terms of a willingness

to test and desire to test products and make sure

that they are as close to gluten-free as they

possibly get. I can't give you a definitive

comment on that. I haven't made any surveys.

CHAIRMAN DURST: Any further questions or

discussion?

Yes?

DR. CALLERY: Pat Callery. Thank you for

110

the review of the relevant biochemistry. Could you

relate the transglutaminase substrate specificity

to these various glutamine-containing --

(Simultaneous discussion.)

DR. KASARDA: Not personally, but other

people have. There are certain sequences that are

susceptible to deamidation and probably

transamidation as well. These have been described

in some recent publications. There are many sites

in the gliadins that are susceptible to

transglutaminase.

DR. CALLERY: The transglutaminase I

understood was an important feature in binding

these proteins and causing the --

(Simultaneous discussion.)

DR. KASARDA: Well, you know, in the MHC

proteins there is a positive charge in the binding

pocket that binds well to a negatively charged

glutamic acid. This does enhance the strength of

the binding to the binding site of DQ2/DQ8.

Now, I didn't get into it, although

possibly someone else will, there seems to be two

111

legs to the celiac disease situation. There is the

adaptive immune system, which has been worked on

quite a bit in terms of this presentation of

gliadin peptides to T-cells, to the T-cell

receptor, and stimulation along that leg.

However, one of the peptides I described

that Mike Marsh had studied, that particular

peptide is not immunoactive. It does not stimulate

the T-cells, yet when instilled directly into the

small intestine it produced changes that were

characteristic of celiac disease.

In the last couple of years, there has

been an interest in the role of the innate immune

system in possibly triggering the first leg of

celiac disease, which then progresses on to involve

the adaptive immune system and the CD4 T-cells of

the lamina propria.

I think that part is becoming pretty well

understood. Ludvig Sollid and his co-workers,

while he was on sabbatical at Stanford, they

actually crystalized a DQ2 with the gliadin peptide

in the binding site. They have defined a number of

112

characteristics of the peptides that are important

for binding.

However, I think we still don't understand

a good part of what is active or toxic about these

peptides, and it may have to do with this

triggering and innate immune response. That is

research that is really developing right now.

CHAIRMAN DURST: Soheila.

DR. MALEKI: Soheila Maleki, USDA.

Essentially, the substrate for the transglutaminase

is the same peptide that is presented by the

antigen-presenting cells?

DR. KASARDA: Well, after you deaminate a

particular glutamine, then the binding strate goes

up for the receptor site on the MHC protein.

DR. MALEKI: Essentially, it is the same

substrate, just after deamination --

(Simultaneous discussion.)

DR. KASARDA: Well, there is also the

question of, Why do you have antibodies to the

transglutaminase? This may involve transamination

reactions where you get a binding of gliadin in the

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transglutaminase, and then this triggers the

apparent autoimmune antibodies to transglutaminase.

DR. MALEKI: I see. Well, I just find it

amazing that when you show the lineup of the

peptides, the homology, that you had a two amino

acid difference and went from immunoreactive to

non-toxic. I'm sure by now they probably can

explain that?

DR. KASARDA: No, they can't.

DR. MALEKI: Well, even in the fitting up

to the transglutaminase or to the processing by

antigen-presenting cells?

DR. KASARDA: Well, it is very puzzling,

very interesting. I really can't answer your

question.

DR. MALEKI: Thank you.

CHAIRMAN DURST: Any further discussion?

(No verbal response.)

CHAIRMAN DURST: All right. Thank you,

Dr. Kasarda.

We are now scheduled for a break. We are

just about on schedule, so we will take a brief

114

break and reconvene at 10:45.

(Thereupon, from 10:25 a.m. to 10:45 a.m.,

there was a pause in the proceedings.)

CHAIRMAN DURST: Our first speaker after

the break is Dr. Alessio Fasano, professor of

pediatrics, medicine and physiology and director of

the Mucosal Biology Research Center, Center for

Celiac Research, University of Maryland School of

Medicine.

PROSPECTIVE STUDIES

DR. FASANO: Thanks so much. I've got to

do this. I need really to thank the FDA, who has

been so kind to invite me, but also to be so

sensitive to use Italian candies.

(Laughter.)

DR. FASANO: This is very nice of you

guys, and we appreciate that. I also want to tell

you guys that because of the other speakers, I

decided to reduce a little bit my talk, so a few

slides have been taken out from the handouts to go

straight to the point.

There has been a general perception that

115

this is a quite young disease, in other words,

something that we are dealing with kind of

recently. I want to put this in the right

perspective and give you some of the background to

justify this prospective study to decide what is

threshold of tolerable gluten.

First of all, believe it or not the first

trace of a description of this disease goes back to

the Roman Empire. This is not something that has

happened in the last few years.

(Slide.)

DR. FASANO: Who really put the disease on

the map is this fellow here. Samuel Gee, at the

end of the past century, around 1890, gave an

historical lecture to a place where I had the

privilege to study for a little while at Saint

Bart's Hospital in London.

He really put celiac disease on the

"scientific map." I took little sentences here and

there from his lectures to give you the sense of

how this guy got the story right more than 120

years ago.

116

He described these as a chronic

indigestion that is met in every single age.

Again, our misconception in the past was celiac

disease was confined to a specific age group. He

knew already that was not the case; it can affect

at any age.

Of course, it is particularly more

frequent in all kids between one and five years

old, and that was the observation at the time. He

spent time and effort to clarify the fact that

everybody can be affected.

Now, symbiotics, hands-on, was the way to

do a disease diagnosis at that time. We didn't

have a lot of sophisticated tools, so it was really

hands-on.

For a gastroenterologist, dealing with a

problem like that means describing feces, stools,

and that's what it is. He introduced with this

description a very important concept about celiac

disease in terms of classical GI presentation,

malabsorption.

In other words, right before they would

117

know about the genetics, right before they would

know about the grains, the eyes are telling us that

the feces are loose, malformed, but not watery,

definitely more bulky than the food taken seems to

account for, i.e., malabsorption.

What is remarkable is this part here. He

ventured also to understand what was the

pathogenesis of the disease and introduced two

concepts: the genetics and environmental trigger.

He said kids that suffer from it are not

all weak in constitution, errors in diet. I want

to clarify that the first time that the link

between celiac disease and grains was made was soon

after World War II. Until then we had no clue

whatsoever what was the trigger leading to celiac

disease.

The link was made during World War II

because there was a higher rate of mortality among

kids in Middle Europe that was not explained.

During World War II, grains were not available

anymore.

They were fed with potato starch, potato

118

flour, and the mortality dropped dramatically, to

reappear after the end of the war when flour was

again available. That is when the link was made.

This guy is already there. Errors in diet

may be perhaps a cause, but whatever. Why, out of

a family of kids all brought up in a much similar

way, should only one suffer?

Again, he is trying to understand what is

the genetic component, what is the environmental

component, why some people have got it and some not

from the same family eating the same stuff.

Then, he finished up by saying, "Okay, I

think that I have a way to get to the bottom line

in treatment. The treatment has to be regulating

food in the main part of the treatment. It is

amazing if you come already with this conclusion.

The allowance of farinaceous food must be small.

Again, I find this remarkable. Highly starchy

food, rice, sorghum, corn-flour are unfit.

Now he is losing himself a little bit when

he says malted food is better. Also, rusks or

bread, provided it is cut thin and well-toasted on

119

both sides, will be all right.

Grant him the benefit of that. I believe

that, again, in 1890 making this kind of statement,

even if he [made] this little boo-boo here, I think

that it is absolutely remarkable.

(Slide.)

DR. FASANO: Now, fast forward that 120

years later, and that is what we understand about

celiac disease. You heard from Dr. Kasarda and

Dr. Murray already that this is an immune-mediated

reaction.

It is not an allergic reaction, but rather

right now we really truly believe that this is an

autoimmune condition. In other words, we are in

the same kind of range as multiple sclerosis, type

1 diabetes, and so on and so forth.

Therefore, as such there are two key

elements to develop the disease: You have to be

genetically susceptible. I'm not going to spend

more time about this DQ2/DQ8, but they are the

docking station, the "eyes," of the autoimmune

system to see the trigger from the environment

120

coming in.

It is unique because the only other

autoimmune disease for which we know everything

specifically is the only autoimmune disease for

which we know the trigger, that is, gluten.

I wish that we had that kind of

information for other autoimmune diseases, for

which we will have a solution. Theoretically, we

have on hand the possibility of treatment of this

disease.

However, I will argue that unless we have

a clear rule of engagement, i.e., a food labeling

bill that will really clearly define what is

"gluten-free," this is a theoretical solution but

very difficult to put in practice.

(Slide.)

DR. FASANO: Again, it is pretty obvious

that you have to have these two ingredients, you

have the genes and you have to have the grains.

When they interplay, you may end up developing

celiac disease.

We heard already that variability in terms

121

of the timing, how long it is going to take, the

outcome in terms of symptoms, and so on and so

forth, is unbearable. However, they are all under

the same kind of umbrella of celiac disease.

What are our treatment options at the

moment? If these two elements are absolutely

necessary to developing the disease, I believe it

is a no-brainer, it is pretty simple, there are

only two solutions.

First, we can remove the genes, and I

don't think that we can do that. We are not quite

there yet anyhow. As Dr. Murray explained, we know

some of them but we don't know all of them. Or,

secondly, we eliminate the grains. Those are the

options that we have available. There is no other

way to turn from this.

Don Kasarda went extensively into this. I

didn't know that he was invited, by the way.

However, the bottom line is the only treatment

right now is strict, lifelong -- as you heard, you

don't grow out of this, so you have got to endure

it for the rest of your life -- avoidance of wheat,

122

rye and barley. The oats story, again, I am not

going to go back because you heard about that.

(Slide.)

DR. FASANO: It is pretty obvious what are

the major sources of gluten. This is the easy part

when you have to deal with the patients freshly

diagnosed. It is easy to say, "You know what? No

bread, pasta, pizza, beer, cookies, muffins, and so

on and so forth.

(Slide.)

DR. FASANO: This is a little bit more

complicated, and that is where I believe a food

labeling bill will help. Of course, it is not

necessary to go and say, "You know what? This

muffin that you buy at the bakery needs a label."

We know that already whether it is gluten-free.

However, this stuff here (showing "Sources

of Gluten" slide) definitely needs a label, some of

them, because it is not clear if they have gluten

or not because they can be processed with or

without gluten.

Gluten is a formidable, extremely cheap

123

biological glue. Don told you the physical,

chemical characteristics of the molecule. The

reason why manufacturers use that is because when

you have two elements of a processed food that does

not stick together, the cheapest way to keep them

together over time is to use gluten. Right now,

the label can see just the nature of flavor but not

gluten, not necessarily so.

Then, there are really the tough ones in

which, this is not even food really, a source of

gluten needs to be considered. I can't

conceptualize enough how many times we've gotten

E-mails of people asking, "Is my husband, who has

celiac disease, going to be sick or whatever," or

the Playdough for the kids in kindergarten, and so

on and so forth. These are elements to keep in

mind that we deal with all the time.

Of course, the big deal is right here --

medications, prescriptions. As for foods,

processed foods, also medication they enjoy gluten

as an additive to keep elements together.

Now, while I was saying adhere to a diet

124

is a pure theoretical no-brainer, but in a

practical sense it is extremely complicated. It is

a chronic intervention that you have to do, and you

have to stick with it with full commitment for the

rest of your life.

Every single individual in this room I am

pretty sure that you have made some commitments

here and there to go on a certain diet or to

exercise or to decide to change your lifestyle. To

keep that constantly for the rest of your life, it

takes a lot of stamina. That is the reality of the

story.

That is true particularly in the American

society in which any chronic illness will require

chronic treatment, whether it be diet or exercise

or medication or whatever, will pose a problem of

compliance. Definitely among different

interventions, a diet compliance can be really a

difficult aspect of treatment.

In my book, food is one of the few joys in

life. How many times do we leave home and go to

work, we drive, we don't think about it, and we

125

find ourselves at work without having to pay

attention to directions, streets, and so on and so

forth? We are used to it.

That is the same with food, we are used to

it. However, that is not the case for celiacs

because they have to think about this over and over

and over again. It will become not a natural,

spontaneous activity in life, but it will become a

very, very demanding operation.

(Slide.)

DR. FASANO: Why don't people stick with

diets? This is a survey that was done in

Upstate New York. This statement, and this is just

to paraphrase something that Cynthia was telling

us:

"If I eat less gluten, I will have less

intestinal damage."

Half of the people say, "You know what? I

really don't have to stay a hundred percent gluten

free. As far as I decrease this, I will have less

problems. I will be all right."

"I've lived this long eating gluten, how

126

much will a gluten-free diet really help me now? I

mean, you know, if it's not been a big deal so far,

why should I just dramatically change my lifestyle?

I've survived so far, I'm not going to die from

it."

"It's not me, that I have to do this.

It's my doctor who should tell me when I need

follow-up testing or whether I need to stick with a

diet, and so on and so forth." One-fourth of the

people say that.

Again, you heard Dr. Murray, that

unfortunately some of the confusion is generated by

the professionals, the healthcare professionals.

They don't know the rule of the game, and,

therefore, they cannot transmit how to play the

game.

It is pretty much the sense that you go to

the doctor as an individual that has to teach you

how to play chess, and this fellow has no clue

whatsoever how to move the pieces. Patients have

to learn how to play chess while playing against a

professional player. How fair is that? It is an

127

ongoing process.

This is the one that disturbed me the

most: "Scientists and doctors still haven't proven

that gluten really hurts them." You know, there is

no clear information that gluten is dangerous to

celiacs, and that is quite disturbing.

(Slide.)

DR. FASANO: What are the current barriers

in compliance? Again, you heard about the emotion

of the person, anxiety. There is a tremendous

reaction when you are diagnosed with a chronic

illness, no matter how you want to put it. Now,

grief and fear and denial are part of the story.

The ability to resist temptation and to be

disciplined on a gluten-free diet is tough. There

are feelings of deprivation. A few years ago I was

with one of the patients, and he got the chance to

drink a gluten-free beer. Soon after he started to

drink the beer, I saw tears coming down his cheeks.

His simple statement was, "I've waited 25 years for

this." Imagine, 25 years to drink again beer.

He was disciplined, and he didn't touch

128

it. However, there are many others, particularly

adolescents, in which that kind of discipline is

really hard to obtain.

This is very much the heart of the

problem, fear generated by inaccurate information.

If we do not have clear ideas, we, as

professionals, and one says black and the other one

says white, and the other one says up and the other

one says down, that creates a lot of confusion and

a lack of trust.

(Slide.)

DR. FASANO: Other barriers to compliance

are of course we live in a society that drives 150

miles an hour, and we don't have the time to seek

to prepare our food to enjoy. My kids consider

that the stove is the microwave. The stove does

not exist.

Cynthia teaches us the fact that the new

generation believes that cooking is just powder

mixed with water, stick it in the microwave, and

the only thing that you've got to do is read how

long should that go on and that's it. What

129

sophistication.

Here, assessing gluten content in food and

label reading is the most compelling change in

lifestyle that these people go through. Right now,

I don't know about you guys, but I don't enjoy food

shopping. I really do not. I tend to go at

midnight when nobody is there, because I want in

and I want out.

That is not an option for celiacs. One

thing that will take you, I don't know, half an

hour will take four or five hours for celiacs

because you've got to read every single label to

the nitty-gritty and make decisions.

Many times now I see people with cell

phones calling an 800-number right there on the

spot saying, "I have your Box XYZ, is this

gluten-free or not?" It is cumbersome.

(Slide.)

DR. FASANO: All of this to come to the

heart of what I'm going to share with you guys.

How much is too much? Unfortunately, I can't

conceptualize and stress enough what Cynthia

130

already said. In biology, the absolute zero does

not exist. If you really do believe that we can

achieve zero as gluten-free, this is a pure

theoretical concept that nobody will ever be able

to achieve.

Assume, just for a moment, that we will

have a sophisticated, super-duper sophisticated,

monoclonal ELISA to really go down to zero. To

manufacture food in that way, people in that

particular factory should be dressed with spray

suits, all antiseptic. A piece of bread will cost

$250, because that is what that level of

sophistication and controlled environments will

take. Consequently, it is impossible.

At the same time we need to give industry,

manufacturers, a parameter of what is tolerable and

what is not. There have been many retrospective

studies that Dr. Collin is going to tell us about,

very few prospective studies because they are

extremely challenging to do right.

This study that I am going to show you the

data of has really been coordinated by

131

Dr. Carlo Catassi, who has been involved in this

kind of topic for the past 15 years. He is a

member of our center, and we have been doing this

in coordination for the past four years.

Why do we need to do this? Because again

this is a long-term, strict gluten-free diet. If

we do a prospective study design, we can answer

questions that a retrospective study was not able

to answer.

How we did this? We did it in a way that

the gluten-free diet, people that come in are

already diagnosed on a gluten-free diet. We are

monitoring this gluten-free diet in a blind fashion

where a given amount of gluten is added to the

diet, then, the clinical, serological and biopsy

evaluation before and after the microchallenge.

The background noise, this is very

important, is caused by possible contamination of

the food was minimized by using a control group, in

other words, to really do this by the book.

(Slide.)

DR. FASANO: Studies done in the past, for

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example, from Dr. Catassi almost a decade ago,

showed a linear relationship between the amount of

gliadin -- that is the toxic part of the story here

-- a daily dose, and it causes damage between 100

and 1,000 milligrams a day.

The intraepithelial lymphocytes -- and we

are going to go back to what these intraepithelial

lymphocytes are all about, the meaning -- was the

most sensitive index, not the serology and not the

symptoms.

What you heard already from Dr. Murray is

that after all these red flags the antibodies may

not be sensitive enough to uncover exposure to

gluten. Indeed, even 10 years ago this was very

clear.

(Slide.)

DR. FASANO: Why do this again? If it was

done 10 years ago, why revisit this if we have

already the information? Several reasons. The

need, first of all, to investigate the effects of

lower gluten doses. Because at that time they were

using large doses, because that was the level of

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sensitivity of the tests for the foodstuff.

There is a need for prolonging the

duration of the microchallenge. In the past, the

longest that we went was a month, and people would

ask, "How about two months or three months?"

How about if the period, the lag period,

between the exposure to gluten and when you react

is longer? You believe it to be safe for one

month, but you keep going, and eventually you

react.

There is a need of a control group that

was never used before, and, most importantly, you

heard that gliadin is part of the story. They are

the glutamines.

If you do the study just as done in the

past, you may really not uncover what is really the

story; in other words, what you leave out there is

not pure gliadin but rather this mixture of

proteins that Don Kasarda was telling us about.

(Slide.)

DR. FASANO: I don't want to spend too

much time on this, but for a matter of

134

quantification, to give a sense of what we are

talking about. In 200 grams of wheat-based

products -- bread, pasta, so on and so forth -- you

heard that the main proteic fraction in wheat is

gluten. For 8 to 14 percent of the overall amount

is wheat. Gluten is 75 percent of all the protein.

Between gluten and glutamine, we can say that all

of this 8 to 14 percent are these toxic proteins

for celiacs. This 8 to 14 percent translates into

15 grams.

The real toxicity, the main toxic, is due

to the gliadins. Again, glutamines contributed to

toxicity. Of the 200 grams, 8 to 14 percent is

equal to 15 grams. Half of it is gliadin. Gliadin

has more than 50 toxic fragments, and so on and so

forth.

If you go on a gluten-free diet, an adult

that is on a gluten-free diet, roughly, consumes --

I mean, in a normal diet, roughly, the amount that

you consume is this, 15 grams. Roughly, you

consume 200 grams of wheat-based products.

If you are on a gluten-free diet, a

135

typical gluten-free diet, the subject consumes

gluten-free flour-based, that is roughly 80 grams.

The key element is how much of this 80 grams of

gluten-free products can be contaminated with the

toxic element, gluten? How much is the amount that

you can tolerate? That is the heart of the problem

here.

(Slide.)

DR. FASANO: That prompted the design of

the study. It is a quite complicated study. The

aim was to evaluate the consequences of the

protracting just minimal intake, either 10 of 50

milligrams, a very small intake.

In a group of adult celiacs on long-term

treatment with the gluten-free diet, why this

amount? Because, again, 100 milligrams was already

tested and proved to be dangerous 10 years ago.

How the study was designed was as a

multicenter, prospective randomized,

placebo-controlled, double-blind and was a

three-year study. It was entirely sponsored by the

Italian Celiac Society.

136

The reason why we did it in Italy, as I

was mentioning before, is mainly because economical

support of such a complicated and expensive study

could be executed at this time only in a place

other than the United States where we don't have

that kind of resources.

(Slide.)

DR. FASANO: Who was eligible? Patients

with biopsy-proven celiac disease had to be on a

gluten-free diet for at least two years. These

people that had been diagnosed with all of the

criteria are accepted and have to be complying with

the diet for at least two years.

If you are younger than 18 years old, poor

compliance, abnormal results at the baseline

evaluation or you have IgE deficiency, that will be

an exclusion criteria.

(Slide.)

DR. FASANO: Now, how we did this? Well,

again, these people were heroes to accept such a

study, but this was the only way to do it. These

people would come in to be scrutinized to see if

137

they were eligible.

If they were eligible, a consent form was

obtained and there was an intense, strict

monitoring of their gluten-free diets for a month

before the beginning of the study was obtained.

Baseline clinical serological and a biopsy was

obtained. In other words, they underwent a

endoscopy with a biopsy to show that they were

fine.

They were blindly randomized in three

groups, either no gluten, 10 milligrams of gluten

or 50 milligrams of gluten. They were followed for

three months. At a monthly interval there was a

check with the serologist for symptoms.

At the end of the study, at the end of the

three months, once again there was a clinical

evaluation and a serological evaluation and a

second intestinal biopsy under endoscopy.

This was the kind of study that this was

the only way, given the fact that the we know

symptoms and serology tests cannot be sensitive

enough to do this right.

138

(Slide.)

DR. FASANO: The purified gluten was used

for the challenge. Gluten -- or lactose-containing

placebo -- capsules were randomly prepared. The

lab tests were centralized. There was monthly

monitoring of adherence to the protocol; it was

checked by a nutritionist.

Measurement of gluten contamination in

commercially available gluten-free food that they

had during the challenge was checked by ELISA. The

serum AGA and anti-tTG antibodies were checked; a

biopsy was performed with morphometry; there was an

intraepithelial lymphocytes count; and control

biopsies from non-celiac patients were used.

(Slide.)

DR. FASANO: These are the foods that they

had a gluten-free foods. You keep in mind that in

Italy right now the food labeling policy is to be

labeled as gluten-free you have to have 20 parts

per million or less.

Indeed, with this simple exception, the

vast majority of the foods that these people there

139

are eating was gluten-free, by definition of the 20

parts per million.

Consequently, the only gluten that these

people were seeing was actually the ones that were

dealing with the challenge, if they were in the

group of gluten exposure.

(Slide.)

DR. FASANO: We were able to recruit 39

people, who were divided equally into three groups.

There were a couple of things that were interesting

to us.

Of all the parameters that we measure, two

are extremely important to establish the health of

the intestine and the exposure to gluten damage,

one was the villous height/crypt depth ratio. It

is very typical use of morphometric analysis that

we do in clinical practice.

Typically, we want to see this: roughly, a

ratio of 3:1. In other words, the height of the

line has to be 3 times the depth of the crypt.

That is what typically we consider to be normal.

Despite the fact that they were on a

140

gluten-free diet, despite that, they fulfilled the

criteria. They were gluten-free, symptom-free,

immunologically negative, and all the 9 yards.

They went on a one-month controlled diet.

When we did the starting biopsy, there was

a slight decrease of the villus-crypt ratio,

meaning, the villi were a little bit shorter. That

is what happens when you have an insult, the villi

become short and the crypts go deeper.

The other parameter is the number of

CD3-positive cells, the intraepithelial lymphocytes

if you wish, was again 20 per hundred entrocytes

and controls and 30 in the celiacs on a gluten-free

diet.

Therefore, at baseline already something

was going on. It is like there is a status of

inflammation in which this is like a very

well-trained athlete, ready to react to anything if

it smells gluten coming through. It is really at

the edge, ready to jump.

There was a strong correlation between the

number of intraepithelial lymphocytes and the

141

villus/crypt ratio, meaning, that the more healthy

is tissue, the less intraepithelial lymphocytes.

The healthier the tissue -- when the crypts are

elongated and the villa get short, the more

intraepithelial lymphocytes are there. The

intraepithelial lymphocytes are really soldiers

that the immune system sensed at the forefront and

ready to fight the battle. That is what it is.

(Slide.)

DR. FASANO: Now, what kind of symptoms

after the three months these people experienced in

the three groups? There were not really

significant differences: abdominal distention,

anemia, iron deficiency, loss of appetite,

bloating, and so on and so forth.

There were equally distributed in all

groups including the placebo, but two really stand

out -- all in the 50 milligrams. This stomatitis

and the mouth, there are the typical signs of

mucosal involvement of the oral cavity in celiacs,

well-described, it was present only in the

50 milligrams. Weight loss was experienced only in

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the 50 milligrams. For the rest, we didn't see any

major differences.

To revisit the concept that the antibodies

were useless -- these are the antibodies, IgA and

anti-tTG and IgG anti-gliadin antibodies -- before

and after the challenge in placebo 10 and 50

milligrams, there was no difference. Pretty much

there was no difference among the groups.

What we saw as the difference was the

villus/crypt ratio, that all in the 50 milligrams

started to decrease to a level of significance.

After three months, we saw the crypts become a

little bit deeper and the villi to become a little

bit shorter. This translates in the fact that

there was damage that started to occur, or possibly

damage that started to occur.

The intraepithelial lymphocytes, there are

these spots here (indicating). Again, these are

lymphocytes under normal circumstances you see in a

smaller quantity in between epithelial cells.

It came to be of a very increased number

in people with 50 but not in 10, not reaching

143

statistical significance, but these are trends that

I have the obligation to report. It is not

significant that there are more of these cells in

the 50 milligrams compared to the starting point,

but it is a trend there.

(Slide.)

DR. FASANO: I believe the heart of all of

this is this table. I believe this really cuts to

the chase. It is extremely confusing, particularly

to patients, when you talk about milligrams and

parts per million. What the heck are you talking

about? Why do we use this parameter of parts per

million and not just straight milligrams?

Because, by the way, say, you do the study

and you show that 50 milligrams could be dangerous,

so how can it be 10 milligrams? How much is 10

milligrams? How much of a pizza is 10 milligrams?

You say, "Well, let me give you the bad news. It's

less than a fraction of a crumb of a piece of

bread. That is what we're talking about.

Still, it doesn't give you clearly what is

the magnitude of the stuff that we are talking

144

about. The reason why we prefer to express in part

per million rather than in milligrams is because

the amount that is tolerable really depends on how

much you eat.

(Slide.)

DR. FASANO: As you see here, this is the

daily intake of gluten-free flour or whatever

products are based on gluten-free. If you eat

50 milligrams, of course you end up to ingest much

less than 300 milligrams of the substance that you

are eating.

Let's say that, for example, we set the

parameter at 200 parts per million. If we want to

accept the outcome of this study as something to

keep in mind, 10 milligrams is safe for everybody,

50 milligrams start to be questionable.

If you set the threshold at 200 parts per

million, if you eat a relatively small amount of

the stuff a day, you are okay. If you eat a little

bit more, you are in an area that we don't quite

know, because again it is between 10 to 50

milligrams. You can argue, "Is 40 okay? Is 30

145

okay?" We don't know.

Definitely, if you eat 300 grams a day --

in other words, you eat large amounts of

gluten-free products that is contaminated to the

level of 200 parts per million -- you start to go

into the red zone. That is dangerous.

If you go down this table, you see that if

you set 20 parts per million, no matter how much

your Italian lifestyle of eating like crazy food

that is gluten-free based, no matter how far you

go, you still are well below the threshold.

Therefore, at least based on this study,

that I believe has been done really the way that it

is supposed to be done, long enough, because three

months is definitely a long period, a threshold of

20 parts per million should be safe for the vast

majority of the people because it will keep you way

below the cutoff that seems to be dangerous, i.e.,

the 10 milligrams.

(Slide.)

DR. FASANO: Now, this litany of names is

just to explain that this was not a trivial study.

146

It was a multicenter study that involved a

tremendous amount of work and a tremendous amount

of dedication of people that have no business to

undergo this, particularly two endoscopies with two

biopsies. However, it is only because of the

dedication and the commitment of these people that

we have an answer and we have a chance to come to

you today with something that is a little bit less

foggy than so far we have had in terms of

prospective studies.

I will stop there, and I will take any

questions that you have.

CHAIRMAN DURST: Thank you very much.

QUESTION AND ANSWER SESSION

CHAIRMAN DURST: Questions?

Margaret.

DR. BRILEY: Margaret Briley. Can you

tell me, I didn't understand, how often did they do

the biopsies? Every month? Every three months?

DR. FASANO: No. No, no, no, that would

kill us if we do it every month. No, the biopsy

was done at the beginning of the study, at the

147

entrance, and at the end of the study, three months

after, the idea being how much insult did you get

in two months.

What was done at intermediate intervals

was a survey of the diet, to make sure that they

were complying with a gluten-free diet, survey

compliance of taking the pill, and the serological

tests for the antibodies. Those were done on a

monthly basis.

DR. BRILEY: On a monthly basis?

DR. FASANO: That's right.

DR. BRILEY: Thank you. That was good.

CHAIRMAN DURST: Erica.

DR. BRITTAIN: Erica Brittain. If I'm

understanding correctly, the conclusion of the

study is that 10 milligrams daily would be safe,

was shown at least to be fairly similar to your

placebo group in this four-month exposure. How

would you know how that would translate to four

decades of exposure?

DR. FASANO: Only with decades of

prospective study. You are a statistician, and you

148

know better than I do that you've got to start

somewhere.

There is no question in my mind that the

only way to do that is like when you put a new drug

on the market, and you go to Phase I and you do 10

people. When you do Phase II, you do 100 people.

You do Phase III, and 10,000 people. Everything is

fine. Ten years later, because millions of people

took it, it may be that something wrong will come

up, a classical example.

I don't have an answer for you. How do I

know in 10 years what's going to happen? But, you

know, we have to have some way to start. I believe

that this study is giving us a parameter, a

justification, a scientific rationale to say,

"Let's start here."

CHAIRMAN DURST: Okay. Soheila.

DR. MALEKI: Soheila Maleki. I was just

wondering, this is probably not directly related to

your topic, I heard earlier mention of wheat flour

and exposure. How much is inhaled exposure

involved in some of these reactions?

149

DR. FASANO: I don't think that anybody

can answer with scientific confidence that inhaling

is or is not a possible port of entry of gluten for

people with celiac disease to react to.

What we know as a fact, an undisputable

fact, is that the intestine is the port of entry,

the key port of entry. I can tell you anecdotally

that we have patients that react to inhalation of

gluten leading to asthma as an allergic reaction to

gluten rather than to celiac disease.

How confident am I that this could be an

alternative to the other route? I'm not really

confident, because I don't think that we have the

scientific proof beyond any reasonable doubt, as we

do with the other route, that it could be a

possibility.

DR. MALEKI: Thank you.

CHAIRMAN DURST: Ciaran.

DR. KELLY: Yes.

Thank you, Alessio, I agree. Thank you

for sharing the data with us, and I agree that at

least it is a basis that we can begin to work from

150

and make some rational approaches to what is best

for our patients with celiac disease.

A couple of questions: The first relates

to the earlier question about the 40-year

experiment. There is one that there is a

20 part-per-million threshold set already in Italy.

Could you comment on how well that is tolerated by

every or the vast majority of celiac patients in

Italy?

DR. FASANO: Actually, it is much more

than that. There are interesting natural

experiments being done. Italy for many years now

reinforced the 20 parts per million. England has

this 20 to a hundred, and so on and so forth.

As far as I can tell you, this is

something that in Italy the food labeling

legislation setting it at 20 parts per million has

been there for 7 years. It has been considered to

be absolutely safe with very sporadical reports of

reactions.

Now, I think it was telling you when you

have a stomachache and you are a celiac, you tend

151

to go that way to the extreme that some people say,

"Today's ache is because I had gluten."

I mean, this is the reality of the story.

But if you want to, statistically speaking, work on

the large numbers, I would say that 20 parts per

million has been proved to be safe.

DR. KELLY: In your study, then, and this

is something that we discussed a lot yesterday in

the context of food allergy and challenge studies,

is there the potential for bias in selection; in

other words, individuals who are highly sensitive,

in terms of symptomatically highly sensitive, to

low levels of gluten would either be afraid or not

choose to enter the study?

DR. FASANO: Absolutely. Absolutely, no

question about it. The reality of the story is

that if you are extremely sensitive to gluten, you

would be less willing to expose yourself to

something that you know is going to harm you.

The point is, What percentage of the

population does that represent? Is it 10 percent,

20 percent, 50 percent, or a fraction of 1 percent

152

of the celiac population?

You know, I'm pretty sure everybody that

is involved in the clinical care of people with

celiac disease has run into people who are

extremely, extremely sensitive to gluten out there.

The exception are people where actually the problem

is the opposite; these are people who can eat

dangerous amounts of gluten and they do not react.

That is a problem.

CHAIRMAN DURST: Dick Durst. Just to

follow up on that, How did you recruit the people

for these studies?

DR. FASANO: The method of recruitment is

a major advantage of the Italian setting is that

there is a single Celiac Society, and they are

extremely committed. What we did was very simple.

They have a national bulletin, both electronically

and on paper, that is read pretty much by the vast

majority of the members of the celiac community.

I believe that we originally asked for 45

volunteers. That is the number that the

biostatistician told us to go for to have a

153

meaningful outcome. We got 470 volunteers, so we

had to turn people down.

CHAIRMAN DURST: Did you at that point

know which ones were the hypersensitive or the more

sensitive versus other and select on that basis at

all?

DR. FASANO: No. The way that these

people were selected was completely random. In

other words, the least that we had every "X," three

or four up -- I don't know, to make the number --

were called to make that unbiased. We really

wanted a representative portion of the population.

This was done by also sex and age.

Yes?

DR. KELLY: Ciaran Kelly again. I do have

one other question, and it has to do with the

interpretation of the data on villus/crypt ratio

and IEL counts in the controls versus the

well-controlled celiacs.

You showed that there was a small

difference at baseline, even though those

individuals were doing well on a gluten-free diet.

154

Your interpretation is that there is an underlying

immune activation. My question is, Is it possible

or likely or relevant that the 20 parts per million

that they are taking is perpetuating that?

DR. FASANO: What I am trying to convey is

the difference is that the recovery -- even if you

are completely, religiously gluten-free -- is not

100 percent. That is what I meant.

I don't know if this is due to an ongoing

immune response. I believe that to not probably be

the case. Because after all, after all with all of

the machinery in the community, these people have

been proved not to go back to normal. Whereas,

again, the fact is that no matter how you push it,

you can't really go back to normal.

I think that the fact that for three

months, even if you were really "touched," so to

speak, you did not react to 10 milligrams. For me

it was a great level of confidence that this is the

way to go -- together again with data with a

retrospective study, that we are going to hear

about in a moment, and on-the-field exercise in

155

Italy.

CHAIRMAN DURST: Jean?

MS. HALLORAN: Another question about the

sample group. When you did the baseline study, how

much variability did you find in the members of

that group?

DR. FASANO: Let me see if I can go back

on this.

MS. HALLORAN: You had two factors that

you looked at, the villus height --

DR. FASANO: Can you put on the slide show

for a second?

(Ms. Sylvia Smith complies.)

DR. FASANO: You will see that there was

MS. HALLORAN: Slide 32.

DR. FASANO: Can you bring me over there,

please?

(Slide.)

DR. FASANO: There is a fair amount of

variability. You see that, and there is some

overlapping at baseline.

156

If you go down -- keep going -- now, if

you can go up to 28, please?

(Slide.)

MS. HALLORAN: It is 32, I think.

DR. FASANO: You want 32? I thought that

you were talking about the variability of the

villus/crypt ratio. Is that what you are talking

about?

CHAIRMAN DURST: Yes.

MS. HALLORAN: Yes.

DR. FASANO: It is a little bit higher

than that.

Can you go higher?

MS. HALLORAN: Ah.

DR. FASANO: Stop here. I need 26.

(Slide.)

DR. FASANO: All right. You see here,

this is the variability. You see here that this is

the variability. These are the single points. If

there was somebody that was high right here

(indicating), and someone like here, these are the

celiacs. There was a continuum, so it is not that

157

there are people here, people there; it is a

continuum.

This is the standard deviation, and this

is the mean. Again, there is some variability but

not huge. There is much more variability in the

intraepithelial lymphocytes -- you can see this

scatter -- that are being monitored.

CHAIRMAN DURST: Suzanne.

DR. TEUBER: Suzanne Teuber. I would

assume, and this may be a completely incorrect

assumption, that in the population that is

following a gluten-free diet strictly, as those you

indicated you recruited, would actually be a subset

of patients who perceive themselves to be very

sensitive, and thus would have a higher motivation

to follow such a diet.

This would bring up in Italy what percent

of patients do comply with the gluten-free diet?

We heard about the extreme difficulties here and

the poor compliance rate. Is it better in Italy?

Would this mean that, perhaps, this population that

you recruited from really might be a good sensitive

158

population?

DR. FASANO: I think, and I'm paraphrasing

Joe Murray on this, that the compliance with the

diet is the results of many factors, some of them

diet there. Education I believe is at the top of

all.

It is not that you feel it to be more

sensitive or less sensitive. If you understand the

facts, if you understand the rules of the game, no

matter how you are perceived as being sensitive or

not sensitive, you know that you can't cheat. You

know that you need to start with that.

If you go to 10 doctors and they say all

the same things, "I'm sorry, you don't have an

alternative," then the level of confidence

increases. We don't have that here. We don't have

it, honestly.

Let's be honest. We have people, doctors,

that will tell you, "You know, you need to go on a

gluten-free diet." These are the teaching sheets

that were printed 20 years ago. "After three

months, go back on a regular diet. You're going to

159

grow out of it." What level of confidence do you

have?

Definitely, a study like here, like this

done here, will have a tremendous amount of bias.

Because who is going to do that? It will be only

the ones that are extremely compliant. The

population in Italy that is compliant -- in Italy?

I should not say in Italy, in Europe --

because they are like 10 or 15 years ahead of us in

this, because the level of awareness has been there

for quite a long time -- is pretty high.

They understand exactly what is at risk.

That is the reality of the story. It is more than

to be the people with high cholesterol, high blood

pressure and to be on medication because there is

much more flexibility there.

These people they understand that if they

don't comply the pay a price, and they do. The

level of frustration, particularly here, is that

they want to do that, the ones that understand the

game, but they can't because there is no way in

their current situation they can comply.

160

CHAIRMAN DURST: Doug.

DR. HEIMBURGER: Doug Heimburger. Would

you go to the next slide, please, after this one?

DR. FASANO: Sure.

(Slide.)

DR. HEIMBURGER: Does this graph include

the controls or only celiac patients?

DR. FASANO: These are only the celiacs.

DR. HEIMBURGER: Just out of interest, did

you test for this correlation in the controls?

DR. FASANO: Yes, it is the same. We put

it all together, yes. There is a strong

correlation. Again, if you conceptualize this

intraepithelial lymphocytes as, again, the first

folks to go there -- just two weeks ago, for

example, there was a paper in science in which they

claimed that the lymphocytes, they are called

gamma/delta, they are able to present antigens.

They can see gluten and they can start the

entire reaction, at least to the adaptive immunity

Th2 response to interferon-gamma, that will

translate in damage, i.e., to make the villi short

161

and the crypt deeper. That makes a lot of sense.

The more you have, the more cytokines you can use,

the more damage you have.

CHAIRMAN DURST: Dr. Fasano, will you be

around for the discussion this afternoon?

DR. FASANO: Yes. Yes, I will.

CHAIRMAN DURST: Because I think maybe we

will stop the questions.

DR. FASANO: I have my candy so I can't

leave you.

(General laughter.)

CHAIRMAN DURST: Okay. We will probably

move on so we don't go too far into the lunch hour.

Our next speaker is Dr. Pekka Collin. He

is a professor at the University of Tampere Medical

School in Finland. He will discuss retrospective

studies.

RETROSPECTIVE STUDIES

DR. COLLIN: Yes, good morning everyone.

I come from Tampere. You probably know where

Finland is and Tampere is a hundred miles north of

our capitol, Helsinki.

162

(Slide.)

DR. COLLIN: We at least in Tampere think

that is the celiac center of Finland, but maybe

somebody disagrees with that. We have a half a

million people around our hospital and now our

clinical prevalence of celiac disease is

approaching 1 percent. I think it is .7 at the

moment, so we have 1,000 patients with celiac

disease. Consequently, we have tried to examine

both the symptoms and the diet.

(Slide.)

DR. COLLIN: I had some specific issues

which I should address at this meeting, and they

are here. I should explain why we carried out our

retrospective analysis of the gluten content in our

gluten-free products; then, also, calculate what is

the significance of daily gluten exposure in this

small amount of gluten; and then, also, to discuss

is there some variability in the sensitivity of

people with gluten intolerance which has been

discussed here already many times. That should

also include patients who are taking with

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starch-based, gluten-free products and who are

taking oats where we have a lot of experience.

(Slide.)

DR. COLLIN: I think that celiac disease

has been described very well by previous speakers,

so I will go straight into the point. However, I

will emphasize that now we are talking about parts

per million or 10 milligrams or 20 milligrams of

gluten intake.

(Slide.)

DR. COLLIN: In real life, if you have 100

patients with celiac disease, I think 90 percent of

them are taking 15 grams of gluten a day because

they do not know that they have celiac disease.

Only 10 out of 100, for instance, in the

U.S.A. I think know that they suffer from celiac

disease. Of the remainder 10, maybe 3 or 4 do not

follow a gluten-free diet strictly because they

don't care, or it is more likely because there are

not enough products when they are eating out or

eating in restaurants, and so on.

I think that is very important, that we

164

have a good choice of products. That is more

important than some parts per million in order to

achieve a good percentage of compliance.

The amount of threshold, I think it

started more than 10 years ago in Europe. The

celiac societies were very, very active in these

respects. From southern countries, some people say

that we are in northern countries poisoning our

people because they know that we are giving them

wheat-starch-based, gluten-free products.

On the other hand, our society, I think

they are very -- I don't find the right word -- but

I admire them because they said, "Please make the

study. Look at what we are now eating. Celiac

patients are the last who will have some extra,

unnecessary dietary restrictions, so please make a

study where you show whether we are now eating

safely or not." I think that was the background

for our so-called "retrospective study."

At that time we were quite relaxed. We

were not afraid that we are poisoning our people,

because we published a study where we showed that

165

in our patients we did not have, in treating

patients we did not have, any extra mortality and

even we did not have any extra risk of malignant

conditions at that time.

Then, we looked at what the Finnish

celiacs are eating. As expected, the majority of

them took wheat-starch-based, gluten-free products.

(Slide.)

We can also see that compliance was very

good. These patients they were invited, after 5 or

10 years on a gluten-free diet they were invited, a

cohort of those patients, both so-called

"sensitive" and not sensitive, and we can see that

only a small percentage of patients had dietary

transgressions. Although there were a few who

daily or twice a week or once a month had dietary

lapses, most people preferred to follow a naturally

gluten-free diet.

We also show that for these patients their

quality of life is good, and they did not have any

additional symptoms compared to the population. As

has been mentioned many times earlier, symptom is

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not a very reliable objective sign of gluten

intolerance.

(Slide.)

DR. COLLIN: This is an example how

symptoms can be misleading. This is maybe a little

bit out of the topic, but I think this is very

interesting.

We ask family doctors to send us all such

patients who spontaneously reported that they get

symptoms after taking wheat or rye. The majority

of them had also on their own account tried to

avoid or withdraw these products from their diets,

and they experienced clear improvement in symptoms.

We thought that many of them had latent or

overt celiac disease, but to our surprise only 10

percent of people with a clear history of

intolerance to gluten had really celiac disease.

Then, there are some which we thought that they

maybe had wheat allergy.

When I was here yesterday I heard about

that. Yes, the diagnosis is so difficult, so I

hope that I don't have to discuss this in more

167

detail.

However, the majority of them, even with

sophisticated methods, they did not have any signs

of celiac disease and probably they have irritable

bowel syndrome. Hence, we cannot trust symptoms

even in the diagnosis of celiac disease.

(Slide.)

DR. COLLIN: Then, of course we have to go

to small bowel biopsy as they did also earlier. We

took a control biopsy after 5 to 10 years from

these patients who had been diagnosed with celiac

disease and who were asked to come to our hospital.

(Slide.)

DR. COLLIN: What we can see here is that

this is the same villous height/crypt depth ratio

which has been measured by, for instance,

Alessio Fasano. Here is our reference value for

people who have no suspicion of celiac disease.

They have come to endoscopy because of suspected

some gastrointestinal disorder, reflux symptoms or

dyspepsia.

We can see that in our long-term treated

168

patients, there is a 95 confidence interval, so it

was exactly the same as in our non-celiac people.

I could show also a similar slide of

intraepithelial lymphocytes, very similar. They

did not have extra intraepithelial lymphocytes.

We did not either have any so-called

"highly sensitive" patients with celiac disease.

We had some here who had not a complete recovery in

the mucosa.

After dietary inspection, it turned out

that all of these people are taking occasionally

gluten. Even once a month, I think that was in the

data, the histological recovery was not complete

Then, we had also here are the celiac patients

where the ratio was of course low.

Then, we had some short-term treated

patients, that means from half year to one year.

We show that the healing was not complete at that

time. From this slide we had two questions.

First, when we have a complete recovery,

are those patients still taking some small amounts

of gluten or are their products complete

169

gluten-free?

The second question was, When we have this

incomplete recovery, does it depend on wheat starch

or gluten contamination or is it normal life in

celiac disease? In other words, would the healing

be better if instead of wheat starch used, the use

of naturally gluten-free products?

To the first issue, Are those products

contaminated which have shown that our people are

doing well and their mucosal is healthy? It was

not surprising that most of naturally gluten-free

products had less than 10 ppm gluten.

However, I think it is very important to

realize that some of the so-called naturally

gluten-free products, they may be contaminated with

gluten, even quite high. All of these were

fulfilling the current European Codex standard.

If we go to the wheat-starch-based,

gluten-free flours, there were two with zero

gluten, and as expected most of them contained

trace amounts of gluten. Two had more than 100,

but the majority has less than 100. That was our

170

idea that maybe we can set the limit to 100 ppm.

When I had this slide and my conclusion in

Europe, one of the representatives of industry said

that he was disappointed because I am talking about

100 ppm, and I should have talked about the limit

of 200 ppm because it is much easier for them.

However, I said that we had too few

products here to assert that 200 ppm would be

recommendable. I think I will remind you that

90 percent of our celiacs have used this product

for 40 years or even more, and we have

biopsy-proven results from that so-called challenge

from 5 to 10 years. The mucosal recovery, as I

said, was perfect.

(Slide.)

DR. COLLIN: We also looked at how much

they did use those flours. Maybe somebody who has

taken gluten-free products can know that they are

not necessarily as good as wheat, baking with

wheat.

Nevertheless, here are how the patients

used these products. There was no difference

171

between wheat-starch-based products or a naturally

gluten-free diet. The average was 80 grams, and

the majority took less than 150 grams as you can

see here. There was no correlation between the

villus damage and the amount of data used of loss.

(Slide.)

DR. COLLIN: From here we come to this

conclusion, which maybe you have seen this kind of

table in Alessio Fasano's presentation. Provided

that we set the limit to 100 ppm, and provided that

each of these products also contained the maximum

amount allowed, when patients are taking 100 grams

of those or 200 grams of those the gluten

contamination is from 10 to 20 milligrams.

If you look at Fasano's results and if you

look at some earlier, small studies -- even the

Catassi study, which was referred to, and some

smaller studies made by Sturgis and so on -- I

think we are very, very safe here at the 100 ppm.

I think also that our clinical experience will show

that the same.

Of course, this is not a prospective

172

study, and we did not have any control group, and,

unfortunately, we did not have many patients who

have clear dietary restrictions, so we cannot make

any statistical analogies between those who are --

what is the word -- cheating with their diet and

who are not. However, I think with this kind of

system, we can treat our patients and have good

compliance.

(Slide.)

DR. COLLIN: If I can, go to the issue

whether patients are more sensitive or

hypersensitive patients with celiac disease. When

we look at those patients, we can see that their

mucosal recovery takes place in a different way in

different people. That has been very well shown in

some challenge studies. Where earlier it was

customary to accept diagnosis, we have once again

to challenge the patients to gluten-free diets and

look at if there will emerge new villus atrophy.

We show that in some cases it took two

months or one month to see a mucosal relapse, but

in some cases it took two or three years. Our

173

record is 15 years. Fifteen years with normal diet

and earlier diagnosed celiac disease, after 15

years a mucosal relapse occurred.

Here we can see that in the short-term

some people do not respond, and you could think

that these might be so-called "hypersensitive." If

we give enough time and the patients are truly

following the gluten-free diet, which means that we

must be really accurate that they do not take wheat

at the same time, I think in the long-term we have

almost complete recovery. We did not have any

so-called "hypersensitive."

I think patients with refractory sprue

they can be very sensitive because they do not

respond at all to celiac disease, but that is a

different issue. It is probable that even zero

gluten would not help them. There is something

wrong in their gut. Probably the diagnosis has

been made too late, and it does not recover any

more. I think that refractory sprue is outside of

the topic of this day.

Also, we were discussing with Peter Chen,

174

when he wrote to "Gastrointestinal Endoscopy" that

complete mucosal recovery is not possible, and we

had a very friendly, friendly discussion in the

pages of that journal. However, we said that it is

possible when we have a good choice of products and

people also outside the home know what celiac

disease is what this means for the patient with a

gluten-free diet.

The second issue in my slide was that

could it be that the mucosal healing would be more

rapid in those who are on a naturally gluten-free

diet than in those who are maintaining

wheat-starch-based, gluten-free products?

Here, we carried out a randomized

prospective study of one year in newly detected

celiac disease patients. If we look at the villus

healing here and here, villous height/crypt depth

ratio, there were no differences between these two

groups. We can also see that in one year, you

cannot achieve the limit of three, which is

considered normal.

Similarly, when we look at intraepithelial

175

lymphocytes, they decreased in a similar way in

both patients. At that time, unfortunately, we

could not measure what was the exact amount of

gluten these patients were taking; we did not have

methods. We can assume they took those same

products which were mentioned in my last slide

which contained trace amounts of gluten but not

more than 100 ppm.

(Slide.)

DR. COLLIN: If I may say some words about

oats. It was in Finland, the first publication.

After that, very soon it was accepted for celiacs

in Finland that they may use oats. At the

beginning we were very careful. We followed up

with them each month and looked at what to do, but

now we do not do it anymore.

We made a question out, too. We sent a

question out to members of the Celiac Society, how

do they appreciate oats. As you can see, they like

about the permission to eat oats.

Almost all said that it is a very

significant part of every day gluten-free diet in

176

terms of tasty and low lost. They even thought

that it is healthy, diversifies the diet, and we

have a good availability in Finland of oat

products. I understand that maybe in some

countries oat is not so important.

Some might say that in Finland they are

not eating good, so maybe people in Italy do not

operate yet in the same manner as in Finland, but

we can discuss it.

(Slide.)

DR. COLLIN: Here are how our people have

now used oats, the majority of patients -- not

great amounts, it is only 20 grams, 15 or 20 grams.

There, most of the studies are about approximately

50 grams, so less than in those randomized studies.

Some people do not prefer oats, and that

is the same thing in people in general not only in

celiac patients. Some of them had stopped, and the

reason is that they had developed symptoms. Some

even got a rash, basically dermatitis

herpetiformis. We do not have any proof that the

reason for stopping would be that they

177

simultaneously had mucosal damage. Usually, the

mucosa is good even though the patient has stopped

the diet.

The rest, in dermatitis herpetiformis, we

also saw that even in patients with no oat diet, so

even they may have a temporary rash. There are

some clinical relapses in patients with dermatitis

herpetiformis also.

It is excellent to study these questions,

because we can change the subjective symptoms quite

rapidly to objective science, count the number of

blisters, for instance.

(Slide.)

DR. COLLIN: We also looked at the quality

of life in patients with oats. Actually, there was

no change, difference, compared to patients with no

oats. This also was a prospective, randomized

study in treating celiac disease.

Interestingly, those patients who were

taking oats, they reported more symptoms of

diarrhea, which was statistically significant.

They also reported more constipation, which was not

178

significant. Even in these patients, we did not

have any mucosal deterioration.

From this we learned that if we start on a

gluten-free diet with oats, we must inform the

patient that "You may have symptoms after this. If

you have symptoms, why continue. But it is

improbable that we have done any harm to your

small-bowel mucosa.

We also saw that those who were taking

oats had a little bit more intraepithelial

lymphocytes, not CD3 lymphocytes, which we have

discussed today, but gamma/delta lymphocytes.

The gamma/delta lymphocytes were a little

bit increased in the oat group. I cannot explain

the reason for that, and that has not been

published elsewhere -- but that is the fact.

(Slide.)

DR. COLLIN: Here are my conclusions to

the questions which I was asked to answer. Maybe I

also specific questions which you have, specific

issues which you have to address in the final

report.

179

If I may say something about the

subpopulation, the most highly sensitive people, I

think such people of course may be, but eventually

they have good mucosal recovery, provided that they

follow a gluten-free diet. The majority of these

highly sensitive patients are probably such people

who have advertent or inadvertent gluten intake.

We can also remember that even if it

happens, the consequences are not disastrous,

because they do not develop an anaphylaxis aspect

as do people with peanut allergy as we heard today.

We can quite easily detect these highly

sensitive, if we after the diagnosis, one year

after the diagnosis, take a small-bowel biopsy and

look at whether there is an improvement in the

mucosal architecture. If there is not, we must

consider that they may be very sensitive, but

usually they do not follow the gluten-free diet.

About the risk of malignant diseases, I

think the whole literature tells that those people

who are at an increase risk of malignant lymphoma,

their diagnosis has been made too late. They

180

already have lymphoma when the symptoms of celiac

disease appear and when they get the diagnosis of

celiac disease, or they have had dietary

transgressions for a prolonged time.

Of over 1,000 patients I have seen during

the 15 years, I have seen one patient who has

developed lymphoma after being 5 or 10 years on an

apparently gluten-free diet. The risk of these

severe complications in those small daily intake is

probably very low. Even our new data show the

same, which is now published only in abstract.

Similarly, the mortality, it depends on

those patients who come to the hospital together

with the diagnosis of celiac disease and later,

usually within six months, we can see that they

also have lymphoma.

(Slide.)

DR. COLLIN: What about the oats? Here I

summarized some studies. Those with plus signs

they are those who have shown that oats have no

adverse effect on the mucosa. I think nearly

almost all of these studies are randomized,

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open-randomized. They have a control group with

non-oat. We have hundred of patients who seem to

tolerate oats.

But I think I would be stupid if I did not

see also those two papers and patients who are

sensitive. I cannot close my eyes from the

results, because Don Kasarda told the data very

convincingly.

I don't know who they are. Maybe there

are some who really develop villus atrophy after

taking oats, but that must be an extremely rare

condition. Because, as you see, we have so many,

many patients who are taking oats, and we have not

seen this phenomenon.

Still, we must be careful, and we must be

careful because patients with oats may develop

symptoms. If everything does not go well, of

course we stop the use of oats. However, we must

be aware of that, that maybe there are some rare

patients where it acts the same as gliadin for most

people with celiac disease.

I don't know whether these, my results and

182

recommendations, can be applied in the United

States but that is how we are doing now. Our

celiac society is very happy because we said that

you can continue with starch-based, gluten-free

products.

Thank you very much.

CHAIRMAN DURST: Thank you.

QUESTION AND ANSWER SESSION

CHAIRMAN DURST: Do we have questions?

Suzanne.

DR. TEUBER: Suzanne Teuber. My question

relates to the applicability of the diet parameters

to the United States dealing with how much

gluten-free flour do people in different parts of

the world ingest, if they were to have the option

of knowing that something was truly, truly

gluten-free.

You talk about 100 parts per million. It

was your data that came up with the 80 grams a day

that people ingest. I'm wondering -- you know, we

are not setting any level here today -- in terms of

United States' folks, I have no idea how that would

183

apply. Would this be a safe level for them? Or,

here, would people be preferring to adjust much

more? Do you have any input on that?

DR. COLLIN: I think there is not much

data on that, how much people really in different

parts of the world are really using wheat or other

flours which may be harmful to patients with celiac

disease.

I think that this is a subject for further

studies. Maybe somebody here knows how much celiac

patients are here using gluten-free flours, but I

don't know. I have not seen any publications about

this issue.

CHAIRMAN DURST: Any other questions?

DR. McBRIDE: Margaret McBride. Did I

understand correctly that gluten-free in Finland

means 100 part per million?

I guess for me, as I'm thinking about it,

maybe part of the difference between the two

studies, aside from the obvious

retrospective/prospective, et cetera, is that in

Italy the gluten-free diet did contain some,

184

although very little at 20 parts per million

gluten, in addition to what was administered.

I don't know if there is an estimation of

how much that would be. I'm also thinking maybe

there is more interest in pasta in Southern Europe

than in Northern Europe.

DR. COLLIN: I think that today we have

given the formal Codex standard which says that

200 ppm is okay, but of course we need to

reconsider that.

I think that in the whole of Europe there

will be two limits, that is the 20 milligram which

can be used in the highly sensitive people, but in

the majority of people it is 100 ppm.

Of course, there is a problem with

labeling, how we should label that. We cannot say

that it is "low gluten," because then people will

use that. That is our problem.

What our recommendation is, is that maybe

the majority of people with celiac disease can

tolerate products which are under the limit of

100 ppm.

185

DR. KELLY: Ciaran Kelly. I wonder in

terms of compliance with the diet and acceptance of

the diet, is there a big difference between 20

parts per million or 100 parts per million from the

perspective of the palatability of the food?

DR. COLLIN: I think the important thing

is, at least the industry in Europe says, that, if

we go to very low level, there are not so many

alternatives for gluten-free products, which again

may result in that general compliance will be worse

than I have shown now.

How the products, how they--? I think

that those wheat-starch products, I think they are

very tasteful. Does it depend on the small

milligrams of gluten or not? I don't know. But,

as can be seen, most of the people are preferring

those products instead of naturally gluten-free.

CHAIRMAN DURST: Anyone else?

(No verbal response.)

CHAIRMAN DURST: If not, thank you,

Dr. Collin.

Our final speaker for this morning is

186

Rhonda R. Kane from the Consumer Safety Office of

CFSAN, FDA, on international perspectives on

gluten-free.

INTERNATIONAL PERSPECTIVES ON GLUTEN-FREE

MS. KANE: Good afternoon. My name is

Rhonda Kane. I am with the Food and Drug

Administration, and I was asked to present

information to the Food Advisory Committee about

how the term "gluten-free" is defined in other

countries and the basis for those definitions.

(Slide.)

MS. KANE: My presentation today will

focus on four examples of international or national

definitions of the term "gluten-free" that apply to

labeled packaged foods.

The first two examples I will be

discussing pertain to Codex Alimentarius and they

include, the first one, Codex Standard 118-1981,

which pertains to the Codex standard for

gluten-free foods that was established in 1981, was

amended in 1983 and is in effect today; and, two,

the Proposed Draft Revised Standard for Gluten-Free

187

Foods at Step 7 that is now under consideration by

the Codex Committee on Nutrition and Foods for

Special Dietary Uses as a replacement for the

current standard.

For ease in my presentation, I am going to

refer the Codex Committee on Nutrition and Foods

for Special Dietary Uses simply as the "Codex

Nutrition Committee."

In the early 1990s, members of the Codex

Nutrition Committee agreed that developments in the

characterization of gluten on studies on gluten

tolerance warranted a revisiting of the current

standard and an updating of it.

The current proposed standard has

undergone several revisions and is now at Step 7 of

an 8-step process pending resolution of certain

issues including what method of detection is going

to be used for gluten and the results of gluten

threshold studies in celiac patients. The Codex

Nutrition Committee will be meeting in November

2005, and will be discussing the proposed standard.

The third example of gluten-free that I

188

will be discussing is found in Canada's Food and

Drug Regulations at Section B.24.018. It became

effective on May 1, 1996.

Lastly, I will review the definitions of

both "gluten-free" and "low-gluten" that are found

in Clause 16 of Standard 1.2.8 of the Australia

New Zealand Food Standards Code, and I will also

discuss the definition of gluten found in Clause 1

of that same standard.

(Slide.)

MS. KANE: The current Codex standard that

is in effect today defines "gluten" as "Those

proteins commonly found in wheat, triticale, rye,

barley or oats to which some persons are

intolerant."

The current standard further defined the

term "gluten-free" to mean that "The total nitrogen

content of gluten-containing cereal grains used in

the product does not exceed 0.5 gram nitrogen per

100 grams of the cereal grains on a dry weight

basis."

(Slide.)

189

MS. KANE: The current standard states

that it does not apply to foods which in their

normal form do not contain gluten. Gluten-free

foods are defined according to two categories,

those that contain the cereal ingredients -- wheat,

triticale, rye, barley or oats or their

constituents, which have been rendered gluten-free

-- or those foods in which any ingredients normally

present that contain gluten have been substituted

by other ingredients that do not contain gluten.

(Slide.)

MS. KANE: In comparison, the Codex

Proposed Draft Revised Standard for Gluten-Free

Foods at Step 7 defines "gluten" to be "The protein

fraction from wheat, rye, barley oats or their

crossbred varieties and derivatives to which some

persons are intolerant and that is insoluble in

water and 0.5 molar solution of sodium chloride."

You will see that in this definition and

in others that are occurring in the proposed

standard information within brackets is intended to

indicate that that information is pending

190

additional discussion at the Codex Nutrition

Committee. Their next session meets in November

2005.

The Proposed Standard also defines the

term "Prolamin" to mean "The fraction from gluten

that can be extracted by 40 to 70 percent aqueous

ethanol." This definition specifically identifies

the prolamins: gliadin from wheat, secalin from

rye, hordein from barley, and avenin from oats.

(Slide.)

MS. KANE: The Proposed Standard also

states that it applies to those foodstuffs and

ingredients which have been especially processed or

prepared to meet the dietary needs of persons

intolerant to gluten.

Therefore, this parameter is similar to

the one for the current standard in that neither of

the two standards, the current and the proposed,

would include foods that are naturally or

inherently free of gluten.

The proposed standard also identifies

three categories of gluten-free foods where their

191

definitions specify certain limits on their gluten

content.

(Slide.)

MS. KANE: In the first proposed category,

gluten-free foods consisting of ingredients which

do not contain any prolamins from wheat or all

Triticum species -- rye, barley, oats -- or their

crossbred varieties cannot have a gluten level that

exceeds 20 parts per million. Again, you will see

that "20 parts per million" is within brackets,

therefore, this number is pending.

(Slide.)

MS. KANE: This proposed definition also

specifically cites three examples of grains within

different species of Triticum, they are: spelt,

kamut, and durum wheat.

Although triticale is not one of the

grains that is identified within the definition by

its name, it is included because it is a crossbred

hybrid of wheat and rye.

In the second proposed category of

gluten-free foods, they are those consisting of

192

ingredients from wheat, rye, barley, oats, spelt or

their crossbred varieties that have been rendered

gluten-free and cannot have a gluten level that

exceeds 200 parts per million. Again, "200 parts

per million" is cited in brackets, and it is

therefore pending.

(Slide.)

MS. KANE: In the third proposed category,

gluten-free foods consisting of any mixture of the

ingredients as described in the previous two

categories, cannot have a gluten level that exceeds

200 parts per million. Again, "200 parts per

million" is cited in brackets and it is pending.

(Slide.)

MS. KANE: Based upon my reading of the

session reports for the Codex Nutrition Committee

and related documents, it appears that the

rationale for including two levels, the 20 and 200

parts per million, in the definition of gluten-free

foods was to accommodate different points of view

of the Codex member countries that thought there

should be a different level of gluten based upon

193

their experience with their populations, what would

be adequately protective.

There were some countries that believed

either the lowest limits of detection or 20 parts

per million would be most protective of those that

are very sensitive to gluten.

Twenty parts per million was considered a

practical limit to make it more feasible for

industry to produce gluten-free foods in that

category.

Other countries believed that the higher

level of 200 parts per million would be

appropriate, because they had experiences with

citizens in their country that had celiac disease

where they had been consuming wheat-starch-based

products for years without harm, and they enjoyed

them.

The 20 parts per million level would

essentially prohibit the inclusion of those

wheat-starch-based products. Therefore, it was a

compromise, the low limit and the high limit, and

they realized they could create some confusion on

194

the part of the consumer.

I also want to point out that the proposed

definition of gluten-free foods specifically cites

that whatever detection method is used it should

have a detection limit of at least 10 parts per

million gluten in the product on a dry weight

basis.

(Slide.)

MS. KANE: The next definition I will

discuss is that found in Canada's Food and Drug

Regulations at Section B.24.018, and it states:

"No person shall label, package, sell or advertise

a food in a manner likely to create an impression

that is a gluten-free food unless the food does not

contain wheat, including spelt and kamut, or oats,

barley, rye, triticale or any part thereof."

(Slide.)

MS. KANE: Canada's definition of

gluten-free prohibits the use of derivatives or

constituents of any of the cited grains.

Therefore, wheat starch would not be allowed in a

product that was labeled gluten-free.

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It is my understanding based upon

communication with staff who work with Health

Canada and the Canadian Food Inspection Agency,

that the definition that Canada is using was

developed using a rule-making process, but they

closely coordinated with the Canadian Celiac

Association in the parameters for this definition.

Canada underwent a rule-making process

similar to the one that we use in the United States

where they reviewed the relevant scientific

literature, they published a proposed rule,

considered comments before it went final, and they

determined that back in the mid-1990s that there

was insignificant or insufficient I should say

scientific evidence to support establishing a level

that would be safe for all celiac patients.

(Slide.)

MS. KANE: In the last definitions of

gluten-free that I will be discussing, in the

Australia New Zealand Food Standards Code, first,

in Clause 1 of Standard 1.2.8 of the "Code," which

I will refer to simply as that rather than

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repeating that long name, it defines "gluten" as

"'The main protein in wheat, oats, barley,

triticale and spelt relevant to the medical

conditions, Coealic disease and dermatitis

herpetiformis.'"

It also defines in Clause 16 of that same

standard the terms "gluten-free" and "low gluten."

(Slide.)

MS. KANE: "Gluten-free" is defined as

those foods that contain no detectable amount of

gluten. They also cannot contain any oats or their

products or any cereals containing gluten that had

been malted or their products. It has to meet all

of those three criteria not just one.

In addition, their Code defines the term

"low-gluten foods" to mean those that contain no

more than 20 milligrams of gluten per 100 grams of

food. Now, although not stated in the Code as

such, this level of gluten is equivalent to 200

parts per million.

(Slide.)

MS. KANE: It is my understanding based

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upon communication with Food Standards Australia

New Zealand's staff that they also underwent a

rule-making process where they proposed these

definitions for "gluten-free" and "low-gluten"

before they went final.

They did a review of the relevant

scientific literature. They considered public

comment, and they also consulted with experts in

the appropriate fields to develop the definitions

that are in effect today.

In addition, the fair trading laws in both

Australia and New Zealand were interpreted as

prohibiting the term "gluten-free" from being used

with any foods that contained any detectible amount

of gluten.

(Slide.)

MS. KANE: Further, the definition of

gluten-free was influenced by a lack of reliable

analytical methods to detect gluten in oats and

malted cereals. Essentially, their definition says

not only no detectible amount of gluten, but no

oats or other products, no malted cereals

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containing gluten and their products because of

this limitation of analytical methods.

(Slide.)

MS. KANE: The Code includes two

definitions, "gluten-free" and "low gluten," to

provide citizens who have celiac disease a choice

between which level of gluten-containing foods they

want to consume based upon their individual gluten

tolerance level and the advice of their healthcare

provider.

In closing, I would like to sincerely

thank the staff that I consulted with at Health

Canada, and the Canadian Food Inspection Agency, as

well as Food Standards Australia and New Zealand.

With that, I will take any questions.

CHAIRMAN DURST: Thank you very much.

QUESTION AND ANSWER SESSION

CHAIRMAN DURST: Does the Committee have a

question or comment?

Erica.

DR. BRITTAIN: Erica Brittain. I guess I

find it appealing the idea of the two levels, just

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as a comment, in the last one you cited. This

might be applicable to the allergy situation as

well.

CHAIRMAN DURST: Okay. Anything else?

Mark.

DR. NELSON: Mark Nelson. Did your

contacts in Australia, New Zealand and Canada give

any indication that they might change their

definitions or their categorizations if there were

more work done on thresholds, if that data based

changed?

MS. KANE: That sort of conversation

didn't occur between me and them, but I would think

because they are government agencies, just like FDA

is, if there were newer information on the horizon,

they would probably consider it. Whether they

would go through the rule-making process and change

it, I guess they would base it on the needs of

their own populations.

DR. NELSON: I guess the opportunity for

-- this is Mark Nelson again -- two categories does

have some attractiveness. I guess at Codex it is

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going to be gluten-free and really gluten-free.

(General laughter.)

CHAIRMAN DURST: Suzanne.

DR. TEUBER: Suzanne Teuber. To your

knowledge in talking with these folks, have their

been any consumer-preference studies or behavior

studies completed or underway with how the celiac

disease patient is using these standards in terms

of their overall intake?

MS. KANE: I don't have personal knowledge

of that. However, the Canadian Celiac Association

is very supportive of Canada's definition of

gluten-free. Because they were instrumental in

helping develop it, so they were very supportive of

it.

CHAIRMAN DURST: Yes.

DR. McBRIDE: Margaret McBride. Do I

understand from your slides about the Codex

proposed changes that the term "gluten-free" would

be applied both to those foods that contained none

of the products in question and are lower than 20

parts per million, and to those foods that are

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wheat-based and gluten has been removed but would

contain up to 200 parts per million? I realize the

numbers are in question.

MS. KANE: Right. They do not apply to

the term. They don't want the term "gluten-free"

to apply to naturally gluten-free foods but those

that have been specially processed or prepared

where the formulation has been controlled.

There is a substitution of ingredients or

a removal of gluten from ingredients. It would

cover categories that are wheat-starch-based. That

is where the 200 parts per million definition is

coming into play.

Member countries did not want

wheat-starch-based products to be excluded from

being called gluten-free, if there was only one

definition of 20 parts per million. That is why

they compromised and had the two levels that would

apply.

DR. McBRIDE: A follow-up. Would I assume

that they would then be called something different,

or would we be expecting the consumer --

202

(Simultaneous discussion.)

MS. KANE: No. Right now, as it stands,

they are saying one definition "gluten-free" to

apply to three categories of gluten-free. However,

that could change.

Now, keep in mind all of this

is pending. It is at Step 7 of an 8-step process.

I know there is a Working Group, the Prolamin

Analysis and Toxicity Group. That information will

come into play. These levels are not definite and

they could change.

If both of those situations or all three

were called gluten-free, then we would have to

expect that the consumer who felt that they were

very sensitive and wanted truly a very low level,

below 20 parts per million, would have to read and

understand the names for the various grains,

et cetera, that would be on the ones where in fact

products that at least one time had contained

gluten were used.

I understand that, and the report I cited

on my second slide, the "ALINORM Report" is the

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latest one, to my knowledge, that contains the

language of the current proposed standard at

Step 7. It doesn't go into those details about how

it might be labeled alternatively or what

additional information it would include. You're

right, it does create confusion. How would you

know if it is 20 parts? How would you know if it

is 200 parts?

That issue was brought up in some related

documents, but it is not found in the latest

session report. However, you're absolutely right.

DR. NELSON: This is Mark Nelson. I just

want to address that question about the Codex

label. There is a separate committee, Codex

Committee on Food Labeling, and these definitions I

would expect would ultimately be referred to the

Codex Committee on Labeling to address the issue

you have just raised about the potential confusion.

CHAIRMAN DURST: Suzanne.

DR. TEUBER: Suzanne Teuber. I also see

an issue about cross-contamination problems with

foods that you wouldn't expect to contain gluten

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and yet might contain contaminants because some of

these, the rules that you are talking about, really

don't address that.

Do you have any information on that, like

say, corn that may be processed in a place that

also has processed wheat? It really would be

beneficial to the consumer if it were to undergo

testing and have a specific label, and yet these

other definitions in other countries don't seem to

cover that all. It would probably just come out

with no statement. Is that a correct

interpretation?

Or, actually maybe, Dr. Nelson--?

DR. NELSON: I think in Europe and Codex

also has a standard for good manufacturing

practices; the Europeans have the equivalent. I

think the issue there would be the responsibility

of the manufacturer to maintain good manufacturing

practices and prevent as much possible that cross

contact.

CHAIRMAN DURST: Marc.

DR. SILVERSTEIN: Marc Silverstein. Would

205

you clarify the categories of foods to which this

would apply? I would like you to, because I'm not

sure I understood the criteria exactly. If a food

has multiple ingredients, does this apply to all of

the ingredients in the food?

This is packaged and labeled food. One or

the major ingredient may be a food which in its

normal form does not contain gluten, yet there

might be other ingredients perhaps mixed in with it

that would.

Would it be that it applies to a labeled

package food which any of the ingredients contain

gluten, or would it be just the major ingredient

does not contain gluten and there might be some

additive or some other component ingredient?

MS. KANE: It is my understanding it would

apply to all ingredients. It would be selectively.

If a packaged food that is labeled gluten-free, it

would have to conform to the proposed. Of course,

again, it is proposed so it is not a done deal.

However, there are categories going back.

Can we go back? Can you reverse it back.

206

It is probably more towards the front. Okay, that

one right there.

(Slide.)

MS. KANE: That is the first category

consisting of ingredients. It doesn't say primary

ingredients. It means ingredients. That is how I

understand it. Keep in mind I've never been a

member of the U.S. delegation to a Codex Committee

meeting. I do not have firsthand knowledge of the

discussions. It is only based on my reading of

their session reports and related documents. The

way that is written I would interpret that to mean

all ingredients. Maybe someone who has attended

the Codex could speak to that?

CHAIRMAN DURST: Mark.

DR. NELSON: Mark Nelson. I think

everybody would interpret that as all ingredients

not just the main ingredients but including the

minor ingredients, flavors, spices, and so on.

I can just talk a little bit about my

experience in the food industry. I have worked

both for packaged goods companies but also

207

suppliers to packaged goods companies.

They look at it very carefully to find out

what the subingredients might be in, say, flavors

or an additive or carriers or something like that.

I can assure you, being a supplier to companies

like Nestle or Kellogg's or Kraft, we have to

provide a fairly substantial dossier to them for

every ingredient we supply them to deal with issues

like allergens and gluten levels as well. The food

industry itself does take this very seriously.

CHAIRMAN DURST: Soheila.

DR. MALEKI: Soheila Maleki. I guess this

is more a question. It seems to me that based on

what we have seen on some of the slides you've

shown today that there really isn't good analytical

method to be able to determine.

For example, the nitrogen content, you

could measure every protein in there and you could

weigh overestimate the amount of gluten. Measuring

gluten in the insoluble water fraction, that seems

to be, again, if you can solubilize it. If you

can't really detect it, okay.

208

DR. NELSON: I'm sorry, you may have to

start over. Sorry about that.

(General laughter.)

DR. MALEKI: It is kind of a question.

Based on this, I don't think there is really an

analytical method that can make you comply to this,

so how does this work? How are they going to

enforce it?

MS. KANE: Keep in mind that the nitrogen

definition of gluten is the current one. They are

proposing it be defined as the protein fraction for

wheat, rye, barley, et cetera, to which persons are

intolerant and it is insoluble in water and a

0.5 molar solution to sodium chloride.

However, there is an analytical method

component of a standard, and that is pending

because they were talking about the R5 Mendez

method, ELISA. They knew that they would have to

have a method that was sensitive enough, reliable,

accurate and would detect the types of proteins

that they are talking about in their definition.

That is going to be, I'm assuming, part of

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the discussion at the next Codex meeting is to

bring that information about the methodology into

play, because those were the two components, the

methodology and threshold levels. Those are the

two areas needed to be worked out, and so I think

that is going to be the crux of the discussion at

the next Codex meeting.

DR. MALEKI: I just wanted to make a

comment as a follow-up.

CHAIRMAN DURST: Oh, okay.

DR. MALEKI: I'm Soheila Maleki. It seems

like the antibodies, the R5 kit again doesn't

detect gluten it detects gliadin. Maybe Steve can

help with that somewhere along the line.

All right, go ahead.

DR. CALLERY: Pat Callery. If the

analytical part can be worked out, which I think it

can. I wonder if there is an analogy here with

caffeine where we have caffeine-free sodas and

such, which we expect to have no caffeine, and

coffee that is decaffeinated that does have

caffeine in it. The word is not very pretty,

210

"deglutinated."

There may be an analogy that says when it

is gluten-free it is truly gluten-free and when it